Subspecific rodent taxa as the relevant host taxonomic level for mammarenavirus host specificity.

Mastomys natalensis-borne mammarenaviruses appear specific to subspecific M. natalensis taxa rather than to the whole species. Yet mammarenaviruses carried by M. natalensis are known to spill over and jump hosts in northern sub-Saharan Africa. Phylogeographic studies increasingly show that, like M. natalensis, small mammals in sub-Saharan Africa are often genetically structured into several subspecific taxa. Other mammarenaviruses may thus also form virus-subspecific host taxon associations. To investigate this, and if mammarenaviruses carried by M. natalensis in southern Africa are less prone to spill-over, we screened 1225 non-M. natalensis samples from Tanzania where many small mammal taxa meet. We found mammarenavirus RNA in 6 samples. Genetic/genomic characterisation confirmed they were not spill-over from M. natalensis. We detected host jumps among rodent tribe members and an association between mammarenaviruses and subspecific taxa of Mus minutoides and Grammomys surdaster, indicating host genetic structure may be crucial to understand virus distribution and host specificity.

Ecology and distribution of Leptospira spp., reservoir hosts and environmental interaction in Sri Lanka, with identification of a new strain.

Leptospirosis is a neglected zoonotic disease and one of the leading causes of zoonotic morbidity and mortality, particularly in resource-poor settings. Sri Lanka has one of the highest disease burdens worldwide, with occasional endemic leptospirosis outbreaks (2008, 2011). Rodents are considered the main wildlife reservoir, but due to a scarcity of studies it is unclear which particular species contributes to bacterial transmission and reservoir maintenance in this multi-host multi-parasite system. Several rodent species act as agricultural pests both in rice fields and in food storage facilities. To unravel the interactions among the small mammal communities, pathogenic Leptospira spp. and human transmission pathways, we collected animals from smallholder food storage facilities, where contact between humans and small mammals is most likely, and screened kidney tissue samples for Leptospira spp. using PCR. Samples were collected in three climatic zones along a rainfall gradient. Pathogenic Leptospira spp. were detected in small mammal communities in 37 (74%) out of 50 sampled farms and 61 (12%) out of 500 collected individuals were infected. The small mammal community was comprised of Rattus rattus (87.6%), Suncus shrews (8.8%), Bandicota spp. (2.8%) and Mus booduga (0.8%). Three pathogenic Leptospira spp. were identified, L. borgpetersenii (n = 34), L. interrogans (n = 15), and L. kirschneri (n = 1). Suncus shrews were commonly infected (32%), followed by B. indica (23%) and R. rattus (10%). L. borgpetersenii strains similar to strains previously extracted from human clinal samples in Sri Lanka were detected in R. rattus and Suncus shrews. L. interrogans was observed in R. rattus only. A single L. kirschneri infection was found in M. booduga. The presence of human pathogenic Leptospira species in an agricultural pest rodent (R. rattus) and in commensal shrews (Suncus) calls for management of these species in commensal settings. Further investigation of the interplay between pathogen and reservoir population dynamics, overlap in geographic range and the extent of spill-over to humans in and around rural settlements is required to identify optimal management approaches.

SARS-CoV-2 surveillance in Norway rats (Rattus norvegicus) from Antwerp sewer system, Belgium.

SARS-CoV-2 human-to-animal transmission can lead to the establishment of novel reservoirs and the evolution of new variants with the potential to start new outbreaks in humans. We tested Norway rats inhabiting the sewer system of Antwerp, Belgium, for the presence of SARS-CoV-2 following a local COVID-19 epidemic peak. In addition, we discuss the use and interpretation of SARS-CoV-2 serological tests on non-human samples. Between November and December 2020, Norway rat oral swabs, faeces and tissues from the sewer system of Antwerp were collected to be tested by RT-qPCR for the presence of SARS-CoV-2. Serum samples were screened for the presence of anti-SARS-CoV-2 IgG antibodies using a Luminex microsphere immunoassay (MIA). Samples considered positive were then checked for neutralizing antibodies using a conventional viral neutralization test (cVNT). The serum of 35 rats was tested by MIA showing three potentially positive sera that were later negative by cVNT. All tissue samples of 39 rats analysed tested negative for SARS-CoV-2 RNA. This is the first study that evaluates SARS-CoV-2 infection in urban rats. We can conclude that the sample of rats analysed had never been infected with SARS-CoV-2. However, monitoring activities should continue due to the emergence of new variants prone to infect Muridae rodents.

Impacts of selective logging on haemosporidian infection and physiological correlates in tropical birds.

Tropical forest degradation affects host-parasite interactions, determining the probability of animals acquiring an infection. The activation of an immune response to fight off infections requires energy and other resources such as antioxidants which may be redirected from growth and reproduction. A key question is how selective logging-the most common form of tropical forest degradation-impacts the prevalence of avian haemosporidian infection and its correlated physiological responses (nutritional and oxidative status markers). We investigated the prevalence of Plasmodium, Haemoproteus, and Leucocytozoon parasites in 14 understorey bird species in lowland, logged and unlogged, old-growth forests of Borneo. Prevalences of infections were similar between selectively logged and unlogged forests. To explore nutritional and oxidative status effects of haemosporidian infections, we examined associations between infections and plasma proteins, plasma triglycerides, and multiple blood-based markers of oxidative status, testing for an impact of selective logging on those markers. Birds infected with Plasmodium showed higher levels of plasma proteins and non-enzymatic antioxidant capacity, and lower levels of plasma triglycerides and glutathione, compared with haemosporidian-free individuals. Conversely, birds infected with Haemoproteus showed no changes in nutritional or physiological markers compared with uninfected individuals. These results indicate higher metabolic and physiological costs of controlling Plasmodium infection, compared with Haemoproteus, possibly due to higher pathogenicity of Plasmodium. Selectively logged forests had no effect on the responses of birds to infection, suggesting that the environmental conditions of degraded forests do not appear to induce any appreciable physiological demands in parasitised birds.

Molecular detection and genomic characterization of diverse hepaciviruses in African rodents.

Hepatitis C virus (HCV; genus Hepacivirus) represents a major public health problem, infecting about three per cent of the human population. Because no animal reservoir carrying closely related hepaciviruses has been identified, the zoonotic origins of HCV still remain unresolved. Motivated by recent findings of divergent hepaciviruses in rodents and a plausible African origin of HCV genotypes, we have screened a large collection of small mammals samples from seven sub-Saharan African countries. Out of 4,303 samples screened, eighty were found positive for the presence of hepaciviruses in twenty-nine different host species. We, here, report fifty-six novel genomes that considerably increase the diversity of three divergent rodent hepacivirus lineages. Furthermore, we provide strong evidence for hepacivirus co-infections in rodents, which were exclusively found in four sampled species of brush-furred mice. We also detect evidence of recombination within specific host lineages. Our study expands the available hepacivirus genomic data and contributes insights into the relatively deep evolutionary history of these pathogens in rodents. Overall, our results emphasize the importance of rodents as a potential hepacivirus reservoir and as models for investigating HCV infection dynamics.

An integrative approach to identify sand fly vectors of leishmaniasis in Ethiopia by morphological and molecular techniques.

BACKGROUND: Ethiopia is affected by human leishmaniasis caused by several Leishmania species and transmitted by a variety of sand fly vectors of the genus Phlebotomus. The sand fly fauna in Ethiopia is highly diverse and some species are closely related and similar in morphology, resulting in difficulties with species identification that requires deployment of molecular techniques. DNA barcoding entails high costs, requires time and lacks reference sequences for many Ethiopian species. Yet, proper species identification is pivotal for epidemiological surveillance as species differ in their actual involvement in transmission cycles. Recently, protein profiling using MALDI-TOF mass spectrometry has been introduced as a promising technique for sand fly identification. METHODS: In our study, we used an integrative taxonomic approach to identify most of the important sand fly vectors of leishmaniasis in Ethiopia, applying three complementary methods: morphological assessment, sequencing analysis of two genetic markers, and MALDI-TOF MS protein profiling. RESULTS: Although morphological assessment resulted in some inconclusive identifications, both DNA- and protein-based techniques performed well, providing a similar hierarchical clustering pattern for the analyzed species. Both methods generated species-specific sequences or protein patterns for all species except for Phlebotomus pedifer and P. longipes, the two presumed vectors of Leishmania aethiopica, suggesting that they may represent a single species, P. longipes Parrot & Martin. All three approaches also revealed that the collected specimens of Adlerius sp. differ from P. (Adlerius) arabicus, the only species of Adlerius currently reported in Ethiopia, and molecular comparisons indicate that it may represent a yet undescribed new species. CONCLUSIONS: Our study uses three complementary taxonomical methods for species identification of taxonomically challenging and yet medically import Ethiopian sand flies. The generated MALDI-TOF MS protein profiles resulted in unambiguous identifications, hence showing suitability of this technique for sand fly species identification. Furthermore, our results contribute to the still inadequate knowledge of the sand fly fauna of Ethiopia, a country severely burdened with human leishmaniasis.

Evaluation of a pan-Leishmania SL RNA qPCR assay for parasite detection in laboratory-reared and field-collected sand flies and reservoir hosts.

BACKGROUND: In eco-epidemiological studies, Leishmania detection in vectors and reservoirs is frequently accomplished by high-throughput and sensitive molecular methods that target minicircle kinetoplast DNA (kDNA). A pan-Leishmania SYBR green quantitative PCR (qPCR) assay which detects the conserved spliced-leader RNA (SL RNA) sequence was developed recently. This study assessed the SL RNA assay performance combined with a crude extraction method for the detection of Leishmania in field-collected and laboratory-reared sand flies and in tissue samples from hyraxes as reservoir hosts. METHODS: Field-collected and laboratory-infected sand fly and hyrax extracts were subjected to three different qPCR approaches to assess the suitability of the SL RNA target for Leishmania detection. Nucleic acids of experimentally infected sand flies were isolated with a crude extraction buffer with ethanol precipitation and a commercial kit and tested for downstream DNA and RNA detection. Promastigotes were isolated from culture and sand fly midguts to assess whether there was difference in SL RNA and kDNA copy numbers. Naive sand flies were spiked with a serial dilution of promastigotes to make a standard curve. RESULTS: The qPCR targeting SL RNA performed well on infected sand fly samples, despite preservation and extraction under presumed unfavorable conditions for downstream RNA detection. Nucleic acid extraction by a crude extraction buffer combined with a precipitation step was highly compatible with downstream SL RNA and kDNA detection. Copy numbers of kDNA were found to be identical in culture-derived parasites and promastigotes isolated from sand fly midguts. SL RNA levels were slightly lower in sand fly promastigotes (ΔCq 1.7). The theoretical limit of detection and quantification of the SL RNA qPCR respectively reached down to 10-3 and 10 parasite equivalents. SL RNA detection in stored hyrax samples was less efficient with some false-negative assay results, most likely due to the long-term tissue storage in absence of RNA stabilizing reagents. CONCLUSIONS: This study shows that a crude extraction method in combination with the SL RNA qPCR assay is suitable for the detection and quantification of Leishmania in sand flies. The assay is inexpensive, sensitive and pan-Leishmania specific, and accordingly an excellent assay for high-throughput screening in entomological research.

Ecology and seasonality of sandflies and potential reservoirs of cutaneous leishmaniasis in Ochollo, a hotspot in southern Ethiopia.

BACKGROUND: Ochollo is a village in southern Ethiopia burdened with cutaneous leishmaniasis (CL), where Phlebotomus pedifer is the only vector for Leishmania aethiopica and hyraxes are confirmed reservoir hosts. A detailed description of the different players of transmission, and the ecology and seasonality of the vector needs to be established in order to accomplish efficient control programs. METHODS AND FINDINGS: Between March 2017 and February 2018, a monthly sandfly collection was carried out in different habitats and records of temperature and humidity were taken. Rodents and hyraxes were trapped in the dry and wet season. All samples were screened for Leishmania kinetoplast DNA (kDNA). Positive samples were further processed for determination of the Leishmania species and the species of the sandfly/small mammal that was found infected. Additionally, the species of 400 sandfly specimens from different habitats and seasons was identified. 17,190 Sergentomyia and Phlebotomus sandflies were caught and showed an overall kDNA prevalence of 2.6%, all were L. aethiopica infections only found in P. pedifer. The overall sandfly and P. pedifer abundance peaked in the dry season and was negatively correlated with the %RH. The kDNA prevalence varied over the months and was negatively correlated with the temperature. Total sandfly abundance did not differ between the sampled habitats, but P. pedifer was the distinct predominant species only in caves. Moreover, significantly more infected sandflies were found in caves. Only 1/192 rodents were kDNA positive, while 20.0% (5/25) of Heterohyrax brucei were found infected. CONCLUSIONS: This study suggests that caves may be a source of multiplication of the infection. If an outdoor control program would be considered, it would be useful to focus on caves in the wet season, when the sandfly abundance is lowest. The captured rodent species appear not important for transmission and the contribution of hyraxes in transmission should be further investigated.

Molecular taxonomy of Crocidura species (Eulipotyphla: Soricidae) in a key biogeographical region for African shrews, Nigeria.

The taxonomy of African shrew species is still unresolved due to their conserved morphology. This also affects knowledge concerning their geographic distribution. In Nigeria, using mitochondrial Cytochrome b gene sequences, we carried out a survey for shrews from the genus Crocidura across various ecological zones to determine taxa that are present and also to assess their phylogeographic structure. Our analyses include 183 specimens collected with Sherman traps from 19 localities around the country. We detected six taxa: Crocidura olivieri lineages II, III and IV, C. hildegardeae, C. jouvenetae, and C. foxi. Among these, C. hildegardeae and C. jouvenetae are reported in Nigeria for the first time. Phylogenetic comparison of our genetic sequences to those generated from other parts of Africa demonstrate that all species in our study, as currently defined, are in need of taxonomic revision. Geographically, Nigeria seems to represent the easternmost boundary for C. olivieri lineage II and C. jouvenetae, and the western distribution limit of C. olivieri lineage IV and C. hildegardeae. The Niger River appears to be the most significant topographical barrier restricting these taxa. This information is vital to preserving the diversity but also managing the epidemiological potential of these small mammals.

Inefficient co-feeding transmission of Borrelia afzelii in two common European songbirds.

The spirochete bacterium Borrelia afzelii is the most common cause of Lyme borreliosis in Europe. This tick-borne pathogen can establish systemic infections in rodents but not in birds. However, several field studies have recovered larval Ixodes ricinus ticks infected with B. afzelii from songbirds suggesting successful transmission of B. afzelii. We reviewed the literature to determine which songbird species were the most frequent carriers of B. afzelii-infected I. ricinus larvae and nymphs. We tested experimentally whether B. afzelii is capable of co-feeding transmission on two common European bird species, the blackbird (Turdus merula) and the great tit (Parus major). For each bird species, four naïve individuals were infested with B. afzelii-infected I. ricinus nymphal ticks and pathogen-free larval ticks. None of the co-feeding larvae tested positive for B. afzelii in blackbirds, but a low percentage of infected larvae (3.33%) was observed in great tits. Transstadial transmission of B. afzelii DNA from the engorged nymphs to the adult ticks was observed in both bird species. However, BSK culture found that these spirochetes were not viable. Our study suggests that co-feeding transmission of B. afzelii is not efficient in these two songbird species.

Shedding dynamics of Morogoro virus, an African arenavirus closely related to Lassa virus, in its natural reservoir host Mastomys natalensis.

Arenaviruses can cause mild to severe hemorrhagic fevers. Humans mainly get infected through contact with infected rodents or their excretions, yet little is known about transmission dynamics within rodent populations. Morogoro virus (MORV) is an Old World arenavirus closely related to Lassa virus with which it shares the same host species Mastomys natalensis. We injected MORV in its host, and sampled blood and excretions at frequent intervals. Infection in adults was acute; viral RNA disappeared from blood after 18 days post infection (dpi) and from excretions after 39 dpi. Antibodies were present from 7 dpi and never disappeared. Neonatally infected animals acquired a chronic infection with RNA and antibodies in blood for at least 3 months. The quantified excretion and antibody patterns can be used to inform mathematical transmission models, and are essential for understanding and controlling transmission in the natural rodent host populations.

High prevalence of Rickettsia typhi and Bartonella species in rats and fleas, Kisangani, Democratic Republic of the Congo.

The prevalence and identity of Rickettsia and Bartonella in urban rat and flea populations were evaluated in Kisangani, Democratic Republic of the Congo (DRC) by molecular tools. An overall prevalence of 17% Bartonella species and 13% Rickettsia typhi, the agent of murine typhus, was found in the cosmopolitan rat species, Rattus rattus and Rattus norvegicus that were infested by a majority of Xenopsylla cheopis fleas. Bartonella queenslandensis, Bartonella elizabethae, and three Bartonella genotypes were identified by sequencing in rat specimens, mostly in R. rattus. Rickettsia typhi was detected in 72% of X. cheopis pools, the main vector and reservoir of this zoonotic pathogen. Co-infections were observed in rodents, suggesting a common mammalian host shared by R. typhi and Bartonella spp. Thus, both infections are endemic in DRC and the medical staffs need to be aware knowing the high prevalence of impoverished populations or immunocompromised inhabitants in this area.

Mixed breeding system in the hermaphroditic land slug Arion intermedius (Stylommatophora, Arionidae).

Theory suggests that hermaphroditic plants and animals should be either entirely outcrossing or entirely selfing. As such, very few hermaphroditic plants and basommatophoran snails have a mixed breeding system. However, reliable estimates of selfing rates are lacking for most hermaphroditic animals. This partly prevents to delineate the relative contributions of the selective factors that determine selfing and outcrossing rates in hermaphroditic animal taxa. Here, we studied the population genetic structure of, and breeding system in, 11 populations of the hermaphroditic land slug Arion intermedius using five polymorphic microsatellite loci. Moreover, genotype frequencies deviated significantly from Hardy-Weinberg equilibrium expectations for most of the loci in all populations suggesting some level of selfing. Estimates of the selfing level s, suggest moderate levels of outcrossing (mean s based on FIS = 0.84; mean s based on the two-locus heterozygosity disequilibrium = 0.20, or with a ML approach = 0.22). Our study therefore suggests that A. intermedius has a mixed breeding system. A re-analysis of allozyme data from another arionid slug ( subgenus Carinarion) indicates that mixed breeding may be more common in arionid slugs than hitherto was assumed. These results seem therefore at variance with current theoretical and empirical predictions and opens perspectives for the study on the evolutionary factors driving mixed breeding systems in animals.

Taxonomic and population genetic re-interpretation of two color morphs of the decollate snail, Rumina decollata (Mollusca, Pulmonata) in southern France.

The hermaphroditic terrestrial snail Rumina decollata has a mixed breeding system with a high prevalence of self-fertilization. In the Montpellier area (France), the species is represented by a dark and a light color morph. Based on allozyme data, both morphs have been reported as single, homozygous multilocus genotypes (MLG), differing at 13 out of 26 loci, but still showing occasional hybridization. Recent DNA sequence data suggest that each morph is a different phylogenetic species. In order to further evaluate this new taxonomic interpretation, the present contribution explores to what extent populations or color morphs indeed consist of single or few MLG. As such it is shown that both morphs are not single, homozygous MLG, but instead reveal a considerable amount of allelic variation and substantial numbers of heterozygous microsatellite genotypes. This suggests that outcrossing may be more prevalent than previously reported. Nevertheless, both morphs maintain a diagnostic multimarker differentiation in the presence of outcrossing in sympatric conditions, implying that they may be interpreted as species under the biological species concept. Finally, our data challenge the idea that simultaneous hermaphrodites should be either strict selfers or strict outcrossers.

Genetic variation in two land snails, Cepaea nemoralis and Succinea putris (Gastropoda, Pulmonata), from sites differing in heavy metal content.

Allozyme variation was determined in two land snail species (Cepaea nemoralis and Succinea putris) from four localities in northern Belgium. In each locality we selected a polluted and a nearby, less-polluted, reference plot. We examined whether (i) genetic variability differed between the polluted and reference plots, (ii) populations from polluted plots experienced recent bottlenecks, and (iii) certain allele or genotype frequencies were associated with the pollution. Our results suggest that (i) about 13% of the genetic differentiation in C. nemoralis and 5% in S. putris was due to differences among polluted and reference plots, (ii) polluted and reference plots had comparable levels of genetic variation, but in C. nemoralis observed heterozygosities were higher in polluted plots, (iii) most plots showed significant evidence for recent bottlenecks, irrespective of the degree of pollution, so that bottlenecks seem poor indicators of pollution-induced stress in land snails, and (iv) mutagenic or pollution-induced modifications did not seem to account for new allozyme variants in polluted sites. The observed patterns of genetic variation may be explained by the action of genetic drift, pollution-mediated selection, restricted gene flow, or a combination of these processes.

Population genetics and identity of an introduced terrestrial slug: Arion subfuscus s.l. in the North-East USA (Gastropoda, Pulmonata, Arionidae).

Several European species of the terrestrial slug genus Arion have been introduced into North America. A case in point is the species complex A. subfuscus s.l. which has become one of the most abundant slug taxa in North America. In Europe this complex consists of at least two cryptic species, viz. A. fuscus and A. subfuscus s.s., the latter of which is further subdivided in five strongly divergent mtDNA lineages (A. subfuscus S1-S5). In order to determine which of these A. subfsucus s.l. taxa are present in the NE USA and in order to assess their population genetic structure, we compared mtDNA, nDNA and allozyme variation between populations from the NE USA and Europe. Our results show that (1) at least A. subfuscus S1 has become successfully established in the NE USA, (2) founder effects are the most likely explanation for the loss of a large amount of molecular genetic variation in populations from the NE USA (i.e. a loss of 96% of the 16S rDNA haplotypes, 67% of the ITS1 alleles and 46% of the alleles at polymorphic allozyme loci), and (3) part of the remaining genetic variation in NE USA populations was probably due to multiple introductions from the British Isles and the European mainland, and the hybrid structure of most of these source populations. Apparently, the extreme loss of molecular genetic variation in this introduced species has not prevented it from successfully establishing and spreading in novel environments.

Molecular systematics of the endemic Leptaxini (Gastropoda: Pulmonata) on the Azores islands.

The Azorean representatives of the Leptaxini (Pulmonata) are single island endemics, where a high-spired shell distinguishes the monotypic genus Helixena from two slightly different low-spired forms within Leptaxis (azorica and caldeirarum type). We studied the evolutionary history of putative taxa and the three shell-types using 12 allozyme loci and sequences of nuclear (ITS-1 and ITS-2) and mitochondrial DNA (COI and 16S rRNA). While little variation was found in both ITS genes, allozyme and mtDNA divergence was among the highest reported for pulmonate land snails. Generally, phylogeographic patterns are indicative of allopatric differentiation via the successive colonization of (younger) islands, while a major role for adaptive evolution is not supported. The azorica shell-type is monophyletic and has no common history with other sympatric shell-types on the same islands. The (ambiguous) position of Helixena sanctaemariae makes Leptaxis paraphyletic on the Azores and possibly also the caldeirarum shell-type. Helixena can therefore not be distinguished as a separate genus on the Azores. Following a lineage-based concept, representatives on all (ancient) islands should be considered distinct species.