Cattle Farming and Plantation Forest are Associated with Bartonella Occurrence in Wild Rodents.

Bartonella spp. are intracellular hemotropic bacteria primarily transmitted by arthropod vectors to various mammalian hosts, including humans. In this study, we conducted a survey on wild populations of sigmodontine rodents, Akodon azarae and Oxymycterus rufus, inhabiting the Paraná River delta region. The study involved eight grids organized in a crossed 2 × 2 design, where four of the grids were exposed to cattle while the other four were not, and four grids were located in implanted forest while the remaining four were in natural grasslands. Our objective was to examine whether the occurrence of Bartonella spp. in rodents was associated with silvopastoral activities (cattle raising associated with timber production) conducted in the region. Additionally, we evaluated the associations between Bartonella infection and other environmental and host factors. We present compelling evidence of a significant positive association between Bartonella prevalence and the presence of implanted forests and cattle. Furthermore, we identified the presence of a Bartonella genotype related to the pathogen Bartonella rochalimaea, infecting both A. azarae and Ox. rufus. These findings suggest that anthropogenic land-use changes, particularly the development of silvopastoral practices in the region, may disrupt the dynamics of Bartonella.

The Role of Molossidae and Vespertilionidae in Shaping the Diversity of Alphacoronaviruses in the Americas.

Bats are reservoirs of diverse coronaviruses (CoVs), including progenitors of severe acute respiratory syndrome CoV (SARS-CoV) and SARS-CoV-2. In the Americas, there is a contrast between alphacoronaviruses (alphaCoVs) and betaCoVs: while cospeciation prevails in the latter, alphaCoV evolution is dominated by deep and recent host switches. AlphaCoV lineages are maintained by two different bat family groups, Phyllostomidae and Vespertilionidae plus Molossidae. In this study, we used a Bayesian framework to analyze the process of diversification of the lineages maintained by Molossidae and Vespertilionidae, adding novel CoV sequences from Argentina. We provide evidence that the observed CoV diversity in these two bat families is shaped by their geographic distribution and that CoVs exhibit clustering at the level of bat genera. We discuss the causes of the cocirculation of two independent clades in Molossus and Tadarida as well as the role of Myotis as the ancestral host and a major evolutionary reservoir of alphaCoVs across the continent. Although more CoV sampling efforts are needed, these findings contribute to a better knowledge of the diversity of alphaCoVs and the links between bat host species. IMPORTANCE Bats harbor the largest diversity of coronaviruses among mammals. In the Americas, seven alphacoronavirus lineages circulate among bats. Three of these lineages are shared by members of two bat families: Vespertilionidae and Molossidae. Uncovering the relationships between these coronaviruses can help us to understand patterns of cross-species transmission and, ultimately, which hosts are more likely to be involved in spillover events. We found that two different lineages cocirculate among the bat genera Molossus and Tadarida, which share roosts and have common viral variants. The bat genus Myotis functions as a reservoir of coronavirus diversity and, as such, is a key host. Although there were some spillovers recorded, there is a strong host association, showing that once a successful host jump takes place, it is transmitted onward to members of the same bat genus.

Heterogeneous taxonomic resolution of cytochrome b gene identification of bats from Argentina: Implications for field studies.

Bats are among the most diverse, widespread, and abundant mammals. In Argentina, 67 species of bats have been recorded, belonging to 5 families and 29 genera. These high levels of biodiversity are likely to complicate identification at fieldwork, especially between closely related species, where external morphology-based approaches are the only immediate means for a priori species assignment. The use of molecular markers can enhance species identification, and acquires particular relevance in capture-release studies. In this study, we discuss the extent of the use of the mitochondrial cytochrome b gene for species identification, comparing external morphology identification with a molecular phylogenetic classification based on this marker, under the light of current bat systematics. We analyzed 33 samples collected in an eco-epidemiological survey in the province of Santa Fe (Argentina). We further sequenced 27 museum vouchers to test the accuracy of cytochrome b -based phylogenies in taxonomic identification of bats occurring in the Pampean/Chacoan regions of Argentina. The cytochrome b gene was successfully amplified in all Molossid and Vespertilionid species except for Eptesicus, for which we designed a new reverse primer. The resulting Bayesian phylogeny was congruent with current systematics. Cytochrome b proved useful for species-level delimitation in non-conflicting genera (Eumops, Dasypterus, Molossops) and has infrageneric resolution in more complex lineages (Eptesicus, Myotis, Molossus). We discuss four sources of incongruence that may act separately or in combination: 1) molecular processes, 2) biology, 3) limitations in identification, and 4) errors in the current taxonomy. The present study confirms the general applicability of cytochrome b -based phylogenies in eco-epidemiological studies, but its resolution and reliability depend mainly, but not solely, on the level of genetic differentiation within each bat genus.

First detection of "Candidatus Rickettsia wissemanii" in Ornithodoros hasei (Schulze, 1935) (Acari: Argasidae) from Argentina.

The aim of this study was to assess the presence of Rickettsia in soft ticks (Acari: Argasidae) collected from insectivorous bats (Chiroptera) in Santa Fe province, Argentina. First, a subset of ticks were mounted in Hoyer's medium to be determined by morphological characters and then confirmed by sequencing the mitochondrial 16S rRNA gene. Also ticks were processed by PCR assays using primers CS-78 and CS-323, which amplify a fragment of the Rickettsia spp. gltA gene. Positive ticks were subjected to a second PCR round with primers Rr190.70p and Rr190.701n of the spotted fever group rickettsiae ompA gene. A phylogenetic analysis was performed with Maximum-likelihood method, and the best fitting substitution models were determined with the Akaike Information Criterion. Five bats of the species Eptesicus diminutus Osgood, 1915, Eptesicus furinalis (d'Orbigny and Gervais, 1847), Eptesicus spp. (Vespertilionidae), and Molossops temminckii Burmeister, 1854 (Molossidae) were parasitized with Ornithodoros hasei (Schulze, 1935) larvae. One E. diminutus ticks' tested positive to "Candidatus Rickettsia wissemanii", a spotted fever group rickettsiae. The association O. hasei -"Ca. R. wissemanii" detected in this study represents the first evidence of a Rickettsia in Ornithodoros ticks in Argentina and the third report of this association in America. Also, this finding constitutes the first record of "Ca. R. wissemanii" in Argentina. Finally, we found for the first time the insectivorous bats E. diminutus and E. furinalis parasitized with O. hasei larvae. These findings add two new hosts and a new location, the southernmost recorded to date, for O. hasei.

Orthohantavirus genotype Lechiguanas in Oligoryzomys nigripes (Rodentia: Cricetidae): New evidence of host-switching.

To identify and predict situations of increased risk of orthohantavirus infection in humans, it is necessary to study the relationships between the virus and its rodent hosts. The present study investigated orthohantavirus infection in an assemblage of wild Sigmodontinae rodents of the Paraná Delta, Argentina, and providing new evidence of host-switching events. Rodents belonging to the species Oxymycterus rufus (n = 187), Akodon azarae (n = 82), Oligoryzomys flavescens (n = 80), Oligoryzomys nigripes (n = 47), Scapteromys aquaticus (n = 38), Deltamys kempi (n = 7) and Holochilus brasiliensis (n = 2) were captured at 4 sampling sites during 20 trapping sessions. Blood samples were analyzed by IgG ELISA and livers by a nested reverse transcription PCR for the diagnosis of orthohantavirus infection. The amplified products of the S and M orthohantavirus genomes were sequenced and analyzed to determine similarities with species of the Orthohantavirus genus. The species of the Oligoryzomys positive to the virus were confirmed by amplifying and sequencing the complete cyt b gene. Of the 443 serum samples analyzed by IgG ELISA, A. azarae presented the highest host-specific prevalence value (10/82, 12.2%) followed by Ol. nigripes (4/47, 8.5%) and Ox. rufus (1/187, 0.5%). All the sero-positive Ol. nigripes (n = 4) were positive to the amplification of the S and M segments of the Lechiguanas genotype (98% nucleotide identity for both segments). This is surprising given that Ol. nigripes has been previously associated with Juquitiba genotype, not Lechiguanas. The latter is generally associated with Ol. flavescens, which in our study were all sero-negative. In addition, the association Ox. rufus - Pergamino genotype found here is, to our knowledge, novel and another potential evidence of host-switching considering that Pergamino has been originally associated with A. azarae. These findings contribute to the building evidence that contradicts the one-genotype-one-reservoir species premise in the association between rodent reservoirs and orthohantaviruses, and supports the hypothesis that the community structure of sympatric host species may contribute to orthohantavirus dynamics.

Tick host specificity: An analysis based on host phylogeny and tick ecological features using Amblyomma triste and Amblyomma tigrinum immature stages.

Here we have evaluated tick host specificity with two different methodological approaches considering Amblyomma tigrinum and Amblyomma triste immatures as targets. Firstly, the Std* index was applied, which considers host phylogenetic relationships weighted by the prevalence of the parasite; followed by generalized linear models to explore associations between different host species and burdens and prevalence of larvae and nymphs, independently. The Std* index showed that A. tigrinum larvae and nymphs infest host species belonging to different orders and classes, respectively; while A. triste immatures fed on hosts that belong to different tribes, showing that both tick species have low specificity and thus, a generalist behavior. When analyzing prevalence and burdens, we found that both tick species infest some hosts more heavily compared with the rest. Even though immature stages of A. triste and A. tigrinum are generalists, the level of infestation within the range of usual hosts of these two species is uneven. This shows that a generalist behavior may result in dissimilar levels of infestation across a range of usual hosts.

[Amblyomma triste and Amblyomma tigrinum (Acari: Ixodidae) in sympatry in Santa Fe Province, Argentina]. / Amblyomma triste en simpatría con Amblyomma tigrinum (Acari: Ixodidae) en la Provincia de Santa Fe, Argentina.

The aim of this communication is to report, for the first time, the occurrence of Amblyomma triste in Santa Fe province, Argentina, and to add a new isolation place for Amblyomma tigrinum. Both species of ticks are vectors of Rickettsia parkeri, a spotted fever group rickettsia. Ticks were recovered from tourists in August 2014 and December 2015 at the Federico Wildermuth Foundation (31° 59'S, 61° 24'O), San Martin Department, Santa Fe province. Five adult ticks were morphologically identified as A. tigrinum (3 females and 1 male) and A. triste (1 female). This is the first finding including both Amblyomma maculatum group species, A. triste and A. tigrinum, together in the same locality in Argentina. This finding suggests that this site might have favorable features for the development of both species of R. parkeri vector. Further studies including sampling of a larger number of ticks and detection of R. parkeri DNA are needed to better document the epidemiology of this rickettsia in Santa Fe.

Amblyomma triste en simpatría con Amblyomma tigrinum (Acari: Ixodidae) en la provincia de Santa Fe, Argentina / Amblyomma triste and Amblyomma tigrinum (Acari: Ixodidae) in sympatry in Santa Fe Province, Argentina

El objetivo del presente trabajo es comunicar el hallazgo de Amblyomma triste por primera vez en la provincia de Santa Fe, Argentina, y sumar una nueva localidad para Amblyomma tigrinum en la misma provincia. Ambas especies de garrapatas son reconocidos vectores de Rickettsia parkeri, agente etiológico de la fiebre manchada. Los especímenes fueron colectados durante el mes de agosto de 2014 y diciembre de 2015 a partir de turistas en la Fundación Federico Wildermuth (31° 59'S, 61° 24'O), departamento de San Martín, provincia de Santa Fe. Se colectaron 5 garrapatas adultas que fueron identificadas morfológicamente como A. tigrinum (3 hembras y 1 macho) y A. triste (1 hembra). El presente hallazgo de A. triste y A. tigrinum representa el primero en incluir a ambas especies del complejo Amblyomma maculatum en una misma localidad y permite alertar sobre la presencia de vectores de R. parkeri en la zona. Nuevos estudios que incluyan el muestreo de un mayor número de garrapatas y la detección de R. parkeri permitirán dilucidar más aspectos sobre la situación de esta rickettsia en Santa Fe.

The aim of this communication is to report, for the first time, the occurrence of Amblyomma triste in Santa Fe province, Argentina, and to add a new isolation place for Amblyomma tigrinum. Both species of ticks are vectors of Rickettsia parkeri, a spotted fever group rickettsia. Ticks were recovered from tourists in August 2014 and December 2015 at the Federico Wildermuth Foundation (31° 59'S, 61° 24'O), San Martin Department, Santa Fe province. Five adult ticks were morphologically identified as A. tigrinum (3 females and 1 male) and A. triste (1 female). This is the first finding including both Amblyomma maculatum group species, A. triste and A. tigrinum, together in the same locality in Argentina. This finding suggests that this site might have favorable features for the development of both species of R. parkeri vector. Further studies including sampling of a larger number of ticks and detection of R. parkeri DNA are needed to better document the epidemiology of this rickettsia in Santa Fe.

Ecology of the interaction between Ixodes loricatus (Acari: Ixodidae) and Akodon azarae (Rodentia: Criceridae).

The present study explores associations of different factors (i.e. host parameters, presence of other ectoparasites and [mainly biotic] environmental factors) with burdens of Ixodes loricatus immature stages in one of its main hosts in Argentina, the rodent Akodon azarae. For 2 years, rodents were trapped and sampled monthly at 16 points located in four different sites in the Parana River Delta region. Data were analysed with generalized linear mixed models with a negative binomial response (counts of larvae or nymphs). The independent variables assessed were (a) environmental: trapping year, presence of cattle, type of vegetation, rodent abundance; (b) host parameters: body length, sex, body condition, blood cell counts, natural antibody titers and (c) co-infestation with other ectoparasites. Two-way interaction terms deemed a priori as relevant were also included in the analysis. Most of the associations investigated were found significant, but in general, the direction and magnitude of the associations were context-dependent. An exception was the presence of cattle, which was consistently negatively associated with both larvae and nymphs independently of all other variables considered and had the strongest effect on tick burdens. Mites, fleas and Amblyomma triste were also significantly associated (mostly positively) with larval and nymph burdens, and in many cases, they influenced associations with environmental or host factors. Our findings strongly support that raising cattle may have a substantial impact on the dynamics of I. loricatus and that interactions within the ectoparasite community may be an important-but generally ignored-driver of tick dynamics.

Factors affecting patterns of Amblyomma triste (Acari: Ixodidae) parasitism in a rodent host.

Here we offer a multivariable analysis that explores associations of different factors (i.e., environmental, host parameters, presence of other ectoparasites) with the interaction of Amblyomma triste immature stages and one of its main hosts in Argentina, the rodent Akodon azarae. Monthly and for two years, we captured and sampled rodents at 16 points located at 4 different sites in the Parana River Delta region. The analyses were conducted with Generalized Linear Mixed Models with a negative binomial response (counts of larvae or nymphs). The independent variables assessed were: (a) environmental: trapping year, season, presence of cattle; type of vegetation (natural grassland or implanted forest); rodent abundance; (b) host parameters: body length; sex; body condition; blood cell counts; natural antibody titres; and (c) co-infestation with other ectoparasites: other stage of A. triste; Ixodes loricatus; lice; mites; and fleas. Two-way interaction terms deemed a priori as relevant were also included in the analysis. Larvae were affected by all environmental variables assessed and by the presence of other ectoparasites (lice, fleas and other tick species). Host factors significantly associated with larval count were sex and levels of natural antibodies. Nymphs were associated with season, presence of cattle, body condition, body length and with burdens of I. loricatus. In most cases, the direction and magnitude of the associations were context-dependent (many interaction terms were significant). The findings of greater significance and implications of our study are two. Firstly, as burdens of A. triste larvae and nymphs were greater where cattle were present, and larval tick burdens were higher in implanted forests, silvopastoral practices developing in the region may affect the population dynamics of A. triste, and consequently the eco-epidemiology of Rickettsia parkeri. Secondly, strong associations and numerous interactions with other ectoparasites suggest that co-infestations may be more important for tick dynamics than has so far been appreciated.

In vitro isolation and infection intensity of Rickettsia parkeri in Amblyomma triste ticks from the Paraná River Delta region, Argentina.

In the present study, we report the first in vitro isolation and infection intensity of Rickettsia parkeri in Amblyomma triste ticks from Argentina. No genetic differences in the molecular targets evaluated were found between R. parkeri isolates from Argentina and those R. parkeri isolates reported in Uruguay and Brazil, both obtained from A. triste. Only a minor difference was observed when compared to R. parkeri isolated from Amblyomma maculatum from United States. Moreover, the prevalence of infection by R. parkeri in ticks collected from the vegetation in the Paraná Delta was high (20.4%). Interestingly, the distribution of R. parkeri infection intensity observed in A. triste ticks was distinctly bimodal, with approximately 60% of the infected ticks presenting high rickettsial loads (3.8×10(5)-4.5×10(7) ompA copies/tick) and the remainder with low rickettsial levels (5.6×10(1)-6.5×10(3) ompA copies/tick). This bimodality in R. parkeri infection intensity in ticks could determine differences in the severity of the disease, but also be important for the infection dynamics of this pathogen. Further research exploring the distribution of rickettsial infection levels in ticks, as well as its determinants and implications, is warranted.

Seasonality of immature stages of Ixodes loricatus (Acari: Ixodidae) in the Paraná Delta, Argentina.

In this study we analyzed seasonal trends of immature stages of Ixodes loricatus parasitizing Akodon azarae (Sigmodontinae) in a locality of the Paraná Delta, Buenos Aires, Argentina. The data encompassed two consecutive years and the analysis included the control of potential confounders using Generalized Linear Mixed Models (GLMM) with a negative binomial response. Larvae had within-year differences in abundance, but the peak months differed drastically between both sampling years, indicating that there was no consistent seasonal pattern. Regarding nymphs, a seasonal pattern was first apparent, but it vanished completely when potential confounders were considered, strongly suggesting that the association with season was spurious and caused by the confounders. Our results showed that the occurrence of immature I. loricatus in its hosts does not follow a distinct seasonal pattern, and highlight the usefulness of controlling for confounding and statistically assessing consistency between years when establishing tick seasonality.