Borrelia encompasses spirochetal species that are part of three well-defined groups. Two of these groups contain pathogens that affect humans: the group causing Lyme disease (LDG) and the relapsing fever group (RFG). Lyme disease is caused by Borrelia burgdorferi s.l., which is distributed in the Northern Hemisphere, and relapsing fevers are caused by Borrelia spp., which are found in temperate and tropical countries and are an emerging but neglected pathogens. In some departments of Colombia, there are records of the presence of Borrelia sp. in humans and bats. However, little is known about the impact and circulation of Borrelia spp. in the country, especially in wildlife, which can act as a reservoir and/or amplifying host. In this context, the objective of our research was to detect and identify the Borrelia species present in wild mammals in the departments of Caldas and Risaralda in Colombia. For morphological detection, blood smears and organ imprints were performed, and molecular identification was carried out through a nested PCR directed on the flagellin B (flaB) gene. A total of 105 mammals belonging to three orders  were analyzed, of which 15.24% (n = 16) were positive for Borrelia. Molecularly, the presence of Borrelia burgdorferi s.s. in lung tissues of Thomasomys aureus and blood of Mus musculus (Rodentia) was detected, with 99.64 and 100% identity, respectively. Borrelia sp. genospecies from a clade branch of a bat-associated LDG sister group were identified in seven individuals of bat species, such as Artibeus lituratus, Carollia brevicauda, Sturnira erythromos, and Glossophaga soricina. Furthermore, two Borrelia genospecies from the RFG in seven individuals of bats  and rodents  were documented. Additionally, the presence of a spirochete was detected by microscopy in the liver of a Sturnira erythromos bat specimen. These results contain the first molecular evidence of the presence of B. burgdorferi s.s. in South America, which merits the need for comprehensive studies involving arthropods and vertebrates (including humans) in other departments of Colombia, as well as neighboring countries, to understand the current status of the circulation of Borrelia spp. in South America.The genus  Borrelia  comprises about 43 recognized species of gram-negative motile spirochetes, ranging in length from 10–40 µm and 0.2–0.5 µm in diameter  from the RFG  were examined. The prevalence of xamined) a. An ind the RFG . Additioythromos b, while flaB gene (~315 pb) were obtained and were deposited in GenBank with accession codes . Seven of these sequences showed identity between 99.19%–99.64% with B. venezuelensis [MG651650] and Borrelia turicatae [MH632129] from the RFG. Two sequences showed an identity of 99.64% and 100% with B. burgdorferi s.s.  from the LDG, and 15 sequences showed an identity between 94.52% and 98.56% with one of the Borrelia genospecies [MT154618] found in Macaregua Cave in Colombia. Furthermore, four allelic variants were found in the Borrelia typing database, one for RFG and three for LDG (Twenty-four (24) partial sequences of the  for LDG .flaB sequences showed that two of the sequences of this study form a monophyletic clade with B. burgdorferi s.s. with a statistical support greater than 92% and a group with the sequences of the same species that were reported in the USA and Canada, with divergences between 0–1.1% , which branches as a sister group of the LDG borreliae and are related to the sequences recorded in Macaregua Cave, Colombia (B. venezuelensis (Brazil) with a statistical support of 83.5% in the ML tree and with genetic distances between 0–1.1%  and Mus musculus (Rodentia: Muridae)  . Overallis study g.B. burgdorferi s.s. in South America, a species that was considered absent in this region of the continent, where the presence of its known vectors is not recorded [B. burgdorferi s.s. [Our results represent the first molecular evidence of the presence of recorded ,75. Neverecorded ,76,77. Frecorded ,78,79,80recorded ; howevereri s.s. .Borrelia sequences recorded from bats in the present study are closely related to genospecies isolated from Carollia perspicillata in the Department of Santander, Colombia [Borrelia restricted to bats, but further studies are needed to help support this hypothesis. The ML reconstruction is congruent with previous reported phylogenetic inferences ,52,80,83Colombia . This reColombia  and JorgB. burgdorferi s.s. in rodents  indicates their potential role as reservoirs for Lyme disease in the country, as has been observed in other rodents in Eurasia and North America [Peromyscus leucopus) have been evidenced to be competent reservoirs for B. burgdorferi s.l. in North America [B. burgdorferi s.l. and other Borrelia species has been reported in other rodents such as murids [Furthermore, the presence of  America ,85. Cric America ,85,86,87 America . A high s murids ,89. MuriBorrelia in the brain of M. altissimus coincides with the neurotropic characteristic of several Borrelia species [Treponema pallidum [Borrelia species are thought to infect the brain to evade the host immune response and may occasionally invade the blood to facilitate bacterial transmission [B. duttonii and B. turicatae, which have detected in the brains of rodents [B. miyamotoi and B. burgdorferi, which were detected in the brain of rodents and a shrew (Sorex maritimensis) [B. burgdorferi complex have been shown to use the liver as a refuge to evade the immune response in long-term infections [The presence of  species ,91, as ismission . This is rodents ,93, and imensis) . Due to imensis) . Additioimensis) . Furtherfections ,97,98.B. burgdorferi s.s. in South America. Likewise, new associations between mammals and Borrelia sp. are presented for the American continent. These findings suggest that the presence of species of the genus Borrelia could be widely distributed in the wildlife of Colombia. However, complementary studies should be carried out to determine the real status of Borrelia spp. in South America and to establish the role played by wild mammals in the maintenance of infections by these spirochetes, as well as their participation in the enzootic and zoonotic cycles of Borrelia sp.Finally, this research provides molecular evidence of the presence of


Elizabethkingia species are antibiotic-resistant microbes. It is a serious bacterial pathogen that can cause the patient to die. Thanks to the technology of sequencing, we can identify different strains of Elizabethkingia species. The Elizabethkingia genus currently comprises 6 species, E. meningoseptica, E. miricola, E. anophelis, E. bruuniana, E. ursingii and E. occul. In the previous study in Taiwan, E. meningoseptica was the major strain that caused infection from 2009 to 2016. But in another reference, we found that E. anophelis became the dominant strain in other countries. Due to this ascertainment, we did an experiment to check the true situation in Taiwan.Elizabethkingia species from bacteraemia patients. And the storage period from January 2018 to June 2020. Then we used the DNA extraction kit to collect the DNA and did the PCR to check whether the target gene was detected or not. Once anoR gene was detected, it would be E. anophelis, if the mengF gene was detected, it would be E. meningoseptica, or if the ureGF gene was detected, it would be E. miricola.Because of the limitation of the tool, in our hospital, we could spread different strains of Elizabethkingia Species by the traditionally identified methods in our bacteria room. That's why we collected the bacteria to our bacteria bank for research. In this study, we collected 65 E. anophelis became the major species , and E. meningoseptica is the second most common species . We didn't find any E. miricola.In this study, we found E. anophelis remains unclear. The case-fatality rate of patients with E. anophelis infection is critically high, ranging from 24% to 60%. To avoid hospital associated infection with E. anophelis, environment cleaning plays an important role. In a recent study, UVC machines can reduce the infection rate, and may be another solution for environment cleaning besides bleach wipes.The transmission route of


Ticks are arthropods that can host and transmit pathogens to wild animals, domestic animals, and even humans. The microbiome in ticks is an endosymbiotic, pathogenic and is yet to be fully understood.Amblyomma scalpturatum  and Amblyomma ovale  ticks were collected from Tapirus terrestris (T. terrestris) captured in the rural area of San Lorenzo Village, and males Amblyomma sabanerae were collected from Chelonoidis denticulate (C. denticulate) of the Gamita Farm in the Amazon region of Madre de Dios, Peru. The Chao1 and Shannon–Weaver analyses indicated a greater bacterial richness and diversity in male A. sabanerae  compared to male A. scalpturatum and A. ovale . Taxonomic analyses identified 478 operational taxonomic units representing 220 bacterial genera in A. sabanerae and 86 operational taxonomic units representing 28 bacterial genera in A. scalpturatum and A. ovale. Of the most prevalent genera was Francisella (73.2%) in A. sabanerae, and Acinetobacter (96.8%) in A. scalpturatum and A. ovale to be considered as the core microbiome of A. sabanerae and A. scalpturatum/A. ovale respectively.Adult male A. sabanerae collected from C. denticulata showed prevalence of Francisella and prevalence of Acinetobacter in male A. scalpturatum and A. ovale collected from T. terrestris. The greatest bacterial diversity and richness was found in males A. sabanerae. This is the first bacterial metagenomic study performed in A. scalpturatum/A. ovale and A. sabanerae collected from T. terrestris and C. denticulata in the Peruvian jungle.We found a high bacterial diversity in male of The online version contains supplementary material available at 10.1186/s12866-022-02717-5. Babesia and Rickettsia respectively, can be identified by microbiological methods such as microscopy and culture in addition to molecular methods such as new generation sequencing techniques (NGS)  compared to the specific bacteria genus in A.scalpturatum/A. ovale [8 (3.5%)] and A. sabanerae ticks [200 (87.3%)]. We suggest that these differences have a behavioral origin related to the host (T. terrestris and C. denticulata) [A. sabanerae collected from C. denticulata change host that are linked to different genus of reptiles compared male A. scalpturatum collected from T. terrestris which are more specific for its host [In addition, a moderate bacterial microbiome was shared between iculata) –13. Thusiculata) . This hyits host , 35, 36.A. scalpturatum/A. ovale was identified as Acinetobacter (96.8%), whose members cause infections at the level of the respiratory, urinary system and wound, in addition this bacterium tends to acquire resistance to various antibiotics and is of importance in Public Health, especially at the hospital level, Acinetobacter has been reported in a metagenomic study in I. persulcatus, I. pavlovskyi, and Dermacentor reticulatus [Rhodococcus (2.5%) the second most abundant genus in A. scalpturatum/A. ovale has the ability to metabolize a large number of substrates and cause pulmonary infections, especially in immunocompromised people [The most prevalent bacterial genus among of iculatus . Rhodocod people .A. sabanerae, Francisella (73.2%) was the most prevalent bacterial genus. Regarding the role of bacteria in ticks, note that nonpathogenic microorganisms present in ticks could cause infections in humans and other animals. For example, ecological studies have shown that Rickettsia, Francisella, and Coxiella, which are considered vertebrate pathogens, can change their pathogenic role and have a mutualistic and symbiotic relationship with ticks [Francisella it is considered as a representative genus of endosymbionts related to pathways for biotin, folic acid, and riboflavin biosynthesis and it is found on rare occasions in some ticks and in the case of F. tularensis as a causal agent of tularemia, a very contagious and life-threatening disease [Coxiella sp. and Francisella sp. are linked to the synthesis of vitamins necessary for the survival of Amblyomma and Rhipicephalus [Rickettsia, and Rickettsiella, have been reported in ticks [Coxiella neither Rickettsia was found.In th ticks . In the  disease , 39, 40. disease . Symbiotcephalus –45. LikeParacoccus, the second most abundant genus (5.4%) in A. sabanerae, is a coccobacillary bacterium that is typically present in a wide range of ecosystems and this bacterium is considered by its diversity of metabolic production in different ecological environments and with biotechnological interest [interest .A. scalpturatum that is found only in South America is a tick that mainly parasitizes T. terrestris. This tick exhibits host specificity so this is the reason for the specificity microbiome, sometimes A. scalpturatum can bite human and is related to transmit Rickettsia [A. scalpturatum was found in T. terrestris.In our case, ckettsia , 12. A. T. terrestris was likely infected by ticks in the jungle and them could infect human due to the proximity of San Lorenzo Village, where livestock farming and hunting are practiced. Previous studies highlight that A. scalpturatum can infect T. terrestris, Pecari tajacu, and humans [A. ovale collected from T. terrestris can parasite many mammals as P. tajacu and can transmit Rickettsia and is found predominantly in sylvatic areas [T. terrestris.d humans , 12, witic areas , 15. In A. sabanerae was collected from C. denticulata. A. sabanerae can parasite different types of reptile as turtle, a previous study showed Rickettsia in A. sabanerae collected from a turtle (Kinosternon sp). In our study A. sabanerae was found in a turtle (C. denticulata) in the Chacra Gamitana Village where the farming is practicing, so the farmer could be parasite by the ticks and infected with some pathogenic bacteria as Rickettsia and Francisella [ncisella , 35, 36.A. scalpturatum/A. ovale collected from T. terrestris and A. sabanerae collected from C. denticulata.According to previous studies, the endosymbiont bacteria of a species of tick vary depending on the ecology and the number of ticks studied . TherefoAmblyomma ticks are not very common studied on the wild host T. terrestris and C. denticulata in the jungle of Madre de Dios-Perú; therefore, we could not collect a larger sample of ticks. Our interest was to study the microbiota of ticks as A. scalpturatum/A. ovale and A. sabanerae that parasitizes T. terrestris and C. denticulata respectively and who live in the Peruvian Amazon.The small number of ticks was justified by the fact that T. terrestris and 10 males’ ticks collected from C. denticulata, which implies a bacterial microbiome representative of a specific circumstance and ecology. Therefore, studies with a greater number of samples could show a greater diversity of species and different percentages of bacterial abundance.Among the limitations of our study is the bacterial microbiome found in 5 males of ticks collected from A. sabanerae collected from C. denticulata showed prevalence of Francisella and prevalence of Acinetobacter in male A. scalpturatum/A. ovale collected from T. terrestris. The greatest bacterial diversity and richness was found in males A. sabanerae. This is the first bacterial metagenomic study performed in A. scalpturatum/A. ovale and A. sabanerae collected from T. terrestris and C. denticulata in the Peruvian jungle. This study lays the foundations for future studies on the importance of the role of the identified bacteria on arthropods and animal and human health.In this study, we found a high bacterial diversity in male of Additional file 1.


Ixodes ricinus were the most frequently collected. Rhipicephalus bursa, Rhipicephalus sanguineus sensu lato (s.l.), Hyalomma marginatum, Hy. lusitanicum, Dermacentor marginatus, D. reticulatus and Haemaphysalis punctata were also found, with adults as the main stage. The number of collected Hyalomma spp. and R. bursa has been progressively increasing over time. Although bites occurred throughout the year, the highest number of incidents was reported from April to July. The distribution patterns of the tick species were different between the north and the south of the region, which was related to cases detected in humans of the pathogens they carried. Adult men were more likely to be bitten by ticks than women. Ticks were most frequently removed from adults from the lower limbs, while for children, they were mainly attached to the head. Epidemiological surveillance is essential given the increase in tick populations in recent years.We conducted a tick surveillance study in northwestern Spain. Nymphs of I. ricinus were the most frequently collected. Rhipicephalus bursa, R. sanguineus s.l., Hy. marginatum, Hy. lusitanicum, D. marginatus, D. reticulatus and H. punctata were also found, with adults as the main stage. The number of collected Hyalomma spp. and R. bursa has been progressively increasing over time. Although bites occurred throughout the year, the highest number of incidents was reported from April to July. The distribution patterns of the tick species were different between the north and the south of the region, which was related to cases detected in humans of the pathogens they carried. Adult men were more likely to be bitten by ticks than women. Ticks were most frequently removed from adults from the lower limbs, while for children, they were mainly attached to the head. Epidemiological surveillance is essential given the increase in tick populations in recent years, mainly of species potentially carrying pathogens causing emerging diseases in Spain, such as Crimean–Congo hemorrhagic fever (CCFH).Ticks transmit a wide diversity of pathogens to a great variety of hosts, including humans. We conducted a tick surveillance study in northwestern Spain between 2014 and 2019. Ticks were removed from people and identified. Tick numbers, species, development stages, the timeline, seasonal and geographical distribution and epidemiological characteristics of people bitten by ticks were studied. We collected ticks from 8143 people. Nymphs of  Ticks are hematophagous parasites distributed worldwide, and they are of great importance from an epidemiological and clinical point of view. They can transmit a wide variety of pathogens, such as viruses, bacteria and protozoa . FurtherIxodes, Dermacentor, Haemaphysalis, Rhipicephalus and Hyalomma) and 54 species have been found, with I. ricinus being the most widely distributed tick [Borrelia burgdorferi sensu lato (s.l.) complex [Ixodes, Dermacentor, Rhipicephalus, Haemaphysalis and Hyalomma, which are potential transmitters of B. burgdorferi sensu lato, several genospecies of Rickettsia, Anaplasma phagocytophilum and the Crimean–Congo virus [Ticks from 896 species are known around the world, and the most prevalent Ixodidae family comprises 702 species in 14 genera. Moreover, ixodid ticks are the main vectors of zoonotic pathogens in Europe , where 5ted tick ,12. Lyme complex . In the I. ricinus was the dominant species, followed by both species of Dermacentor and H. punctata. In the south, in addition to the species mentioned above (except D. reticulatus) we found R. bursa, Hy. marginatum and R.sanguineus, which were very abundant in zones rich in livestock and dogs. Although some species are active all year round, most of them are most active in spring–early summer (April–July) and autumn. A long time has passed since then, and given the emergence of new species of ticks and the introduction of new TBDs, such as Crimean–Congo hemorrhagic fever (CCHF) and DEBONEL (Dermacentor-borne necrosis erythema and lymphadenopathy), it seems essential to re-examine the current situation in this area of Spain. Thus, the purpose of this work is to update data about species of ticks removed from people and their spatial and temporal patterns. This will allow identifying the risk areas, activity peaks and dynamics of these vectors.The association between global warming and the emergence of TBDs is well documented in Europe and Eurasia . The geo2 and is one of the most extensive regions of the European Union. Although there is a marked continental climate in most of the territory, characterized by cold winters and hot summers with short periods of spring and autumn, regional variations in both temperatures and rainfall allow us to distinguish different climatic domains in the region: continentalized Mediterranean in the center, with semi-arid enclaves in some areas, mountain Mediterranean in mountainous areas in the northeast, east and south and Atlantic in the north , an area located in northwestern Spain. It covers a surface of 94,224 kmhe north . This grR. sanguineus (s.l.) ticks, which were identified only to the “group” level as the re-definition of R. sanguineus sensu stricto (s.s.) [Rhipicephalus turanicus specimens were also reported as R. sanguineus s.l. Tick species, developmental stages , sex and feeding degree were recorded, as well as the epidemiological characteristics of the patients  and anatomic location of the attachment of the tick on the patient. A unique individual identification number was assigned to each tick and its corresponding file.During 2014–2019, ticks were collected from people who went to primary healthcare centers and hospital emergency services in Castilla y León for their removal through a program of the Junta de Castilla y León for the prevention and control of tick-borne anthropozoonoses. The ticks were removed from the hosts with tweezers and sent to our laboratory  at room temperature in a container. Each tick was morphologically identified under a binocular lens in terms of life stage and species using taxonomic reference keys ,22,23. Eo (s.s.)  was not https://geocode.localfocus.nl, accessed on 28 February 2022), prior to data curation. The latitude and longitude were then projected to the coordinate reference system ETRS89 to map the distribution of the species of interest through the years and the median temperature in the provinces of the autonomous community of Castilla y León, using pretty breaks as a data classification method. All the maps were constructed in QGIS 3.18.3 “Zürich”  [Information about tick bites was obtained from the database of the center of reference of the Biomedical Research Institute of Salamanca-Research Center for Tropical Diseases at the University of Salamanca. To obtain the geographical coordinates of every tick bite, we geocoded the locations of tick collections using “Batch geocoder for journalists” (ndation) .Absolute frequencies were used to summarize the number of each species and show visual tendencies across years and seasons. Relative frequencies converted into percentages were used to visualize tendencies between the sex and life stages of the collected ticks, and both frequencies were used to show the epidemiological characteristics of people bitten by ticks. ggplot2 [p were adjusted using the false discovery rate and Bonferroni methods, considering values less than or equal to 0.05 as statistically significant.In addition, we searched for signals of displacement in the geographic distribution of the collected ticks. For the visual pattern, we used geographic coordinates of the previous geocode step. The comparative figures were constructed using the package ggplot2 , in the ggplot2 . The KruI. ricinus was the most frequent species infesting humans (52.29%), followed by R. bursa (12.15%), R. sanguineus s.l. (10.38%), Hy. marginatum (9.08%), D. marginatus (8.06%), Hy. lusitanicum (3.79%), D. reticulatus (2.48%) and H. punctata (1.73%). Although this general trend was maintained over the six years of the study, during recent years, there has been a clear increase in Hy. lusitanicum and Rhipicephalus bursa.A total of 8143 ticks were collected over 6 years, of which 8081 were identified to the species level and life stage . The remaining 62 were discarded as their morphological identification was impossible due to the structural and conservation state in which they were received. Ixodid ticks belonging to five genera and eight species were found ; I. riciD. reticulatus and H. punctata), others underwent changes. Hyalomma lusitanicum is a clear example of how a species has increased over time , especially since 2015. Similarly, this occurred with R. bursa, with a huge growth spike in 2019 . Dermacentor marginatus and Hy. marginatum had roughly identical temporal distributions with clear downward trends in 2017 and 2018 before recovering in 2019. Rhipicephalus sanguineus and I. ricinus both had their maximum peak in 2015. Despite this, both species exhibited a downward trend.Regarding the temporal distribution , while sIxodes, where nymphs (60.31%) were collected in much larger numbers  for all the species, while for Hyalomma species, the adults recovered were mainly males (68.20%). Larvae were recovered only from I. ricinus. This composition of females and males did not change throughout the study. The adult stage  was the most frequent in all genera  except f numbers . For Dern = 6303) was reported during spring and summer (April to July) and the lowest in winter (January and early February) . Ixodes ticks were the most frequent and widely distributed species . Rhipicephalus had one activity peak in spring with a decrease in summer (most bites in May and June) and practically disappeared in autumn and winter in the case of R. bursa. For R. sanguineus, despite the decreases in the number of cases, it was still active during these seasons. Both species of Dermacentor were active all year, although they showed pronounced seasonality with two annual peaks of maximum activity in spring and autumn for D. marginatus and autumn and winter for D. reticulatus, with very little or no presence in summer. Most D. marginatum bites were in April and May, with slightly lower peaks in October and March, while most bites by D. reticulatus occurred in December and March, with a smaller peak in April. Haemaphysalis had a very low occurrence and was limited to spring (May and June).The highest number of tick bites  and by year: 2018 compared with 2015 , 2019 compared with 2014  and 2019 compared with 2015 . In the case of the latitude, this was statistically significant only at the group level .Looking at how the distribution of ticks has varied over the years by analyzing the latitude and longitude , we can Data on sex, age and sites of tick bites on people are shown in 2 = 339.90, df = 21, p < 0.05), we observed statistically significant differences between age groups for each of the species, except in the case of D. reticulatus. We removed the greatest number of ticks from the species I. ricinus, R. bursa, D. marginatus, Hy. marginatum and Hy. lusitanicum from the 55+ age group, while R. sanguineus and H. punctata were mostly retired from the children’s age group (0–14).Using the chi-square test . We observed that for all species, the number of ticks removed from men was significantly higher than from women, except in the case of Ixodes ricinus and Hyalomma spp. were removed more frequently from the lower limbs than from other body locations, while R. sanguineus., Dermacentor spp. and H. punctata were mainly fixed on the head. In the case of ticks belonging to the R. bursa species, the main attachment site was the thorax.Ticks were most frequently removed from adults from the lower limbs (29.12% of 3561), and children were mainly bitten on the head (38.25% of 1422). The emergence of novel tick-borne diseases in recent years has made it essential to understand the distribution of tick populations. Although there are already studies on the distribution of ticks in different areas of Spain ,19,28,29Ixodes ricinus was the most predominant and widely distributed species, both spatially and temporally, with a clear dominance over the others, followed by R. bursa. These results coincide with those observed in other studies carried out by our group seventeen years ago [I. ricinus and R. bursa were also the prevalent species. However, data about other ticks have changed over the years. Hyalomma marginatum is currently the fourth most frequently removed species from humans, followed very closely by R. sanguineus s.l, whereas in a previous study (1996–2002), it was ranked fifth behind D. marginatus. Nevertheless, the percentage of recovered ticks belonging to D. marginatus was higher in the 1997–2002 period. The most evident case of a change in distribution patterns and abundance was found for Hy. lusitanicum, which currently accounts for 4.2% of the ticks removed from humans, compared to 0.85% in the previous study. In this sense, keep in mind that ticks of the genus Hyalomma are the main vectors of CCHFV, which is currently considered an emerging or possibly a re-emerging pathogen in Southern Europe. Although the presence of this virus was already reported in Spain in 2010 [Hyalomma ticks observed in this area. All observed changes in both distribution patterns and frequency of ticks are determined not only by biotic factors such as climate or abiotic factors (vegetation), but also by the accessibility of hosts.ears ago , in whic in 2010 , an incr in 2010 ,32. The I. ricinus over the other species is not unique to the study area. The same pattern has been observed in studies carried out in different locations in Spain [I. ricinus, which is also consistent with what was observed in a study previously conducted by our group [The predominance of in Spain ,33,34,35ur group . Our resur group ,37.Although we cannot conclude that ticks have a preference among human hosts according to their sex, of the 7862 participants included in this study, 65.40% were men and 34.59% were women. This preference for males is very similar to that observed by Fernández Soto in 2003 (62% men and 38% women). The fact that this pattern remains the same as it was almost 20 years ago may indicate that the occupational and behavioral habits in terms of outdoor activities among men and women have not changed. Thus, 22.59% of tick bite victims are in the age group of 14 years old or younger, while the 15–35 group has the lowest number of tick bites at 16.94%, in line with Northern Europe .Differences in anatomical sites of attachment were observed for both children and adults. Tick bites occurred most frequently on the lower limbs and thorax in adults and on the head in children. Similar results have been observed in other studies in Western and Northern Europe, with a predominance of bites on the legs in adults and the head and neck in children ,39,40,41Ixodes were detected throughout the year but mostly in spring, autumn and later winter (March), which differed somewhat from previous years in the same study area where adults were biting mainly in autumn and spring but not in winter [Hyalomma is infrequent in winter, with its peak activity in spring and summer, with June and July being the months with the most reported cases for both species. In recent years, we have seen that Hy. marginatum bites are being brought forward to May. The activity pattern of Rhipicephalus has been maintained in northwestern Spain over time, with two peaks in spring and summer, although as with Hyalomma, in the case of R. bursa, there are many reports of bites as early as May. Dermacentor shows a particular seasonality, being practically absent in summer for both species, which could lead us to think that it does not feel comfortable with hot temperatures. Dermacentor marginatus presents a bimodal activity pattern with peaks in spring (April–May) and autumn, and the peak number of bites always in April. In the case of D. reticulatus, bites occur similarly in autumn, winter and spring (from October to March). This differs from the situation observed in Belgium, where the highest peaks of activity occur in spring (March–May) and late summer and autumn (August–November) [Haemaphysalis is practically limited to spring. Although previous studies have reported cases in summer, we have not seen them.Although ticks are removed during all months of the year, the highest number of tick bites are recorded during spring and summer, with a peak of tick activity in June and July, as observed in this same area in previous studies. These data are also in accordance with several studies carried out in Europe ,42,43,44n winter . These an winter ,36,42,45Dermacentor marginatus, D. reticulatus and H. punctata were mainly distributed in the north of the study area, although the greatest increase in D. marginatus was seen in the southwest. On the other hand, Hy. marginatum, Hy. lusitanicum and R. bursa were found in greater numbers in the south. Hyalomma marginatum is expanding its habitat to southwestern areas, and Hy. lusitanicum in recent years to arid or semi-arid areas in the west. The wider distribution of Hyalomma in the south could explain the increasing occurrence of CCFH virus observed in this area in recent years. Finally, I. ricinus and R. sanguineus were distributed in both areas. However, the greatest number of specimens was found in the northeast for both, especially I. ricinus.While studying the geographical distribution of the ticks, we observed quite stable and different distribution patterns between the north and the south. These distribution patterns were determined by various climatic (temperature and humidity) and ecological factors. I. ricinus nymphs. Ticks bite particularly on the legs of adults and on the head of children. Therefore, although the peak season for tick bites is spring and summer, tick bites are becoming increasingly frequent in autumn and even winter. Seasonal tick patterns have changed in recent years, both in Spain and elsewhere in Europe, where many species of ticks have expanded their distribution. The increased period of activity increases the likelihood of being bitten and therefore of being infected by a tick-borne pathogen. This study is essential for proper epidemiological surveillance. Moreover, knowledge of tick populations and human exposure to tick bites could suggest ways to reduce the risk of tick-borne diseases.Our results show that humans from northwestern Spain are mainly bitten by


The Plesiosauria (Sauropterygia) are secondary marine diapsids. They are the only tetrapods to have evolved hydrofoil fore- and hindflippers. Once this specialization of locomotion had evolved, it remained essentially unchanged for 135 Ma. It is still controversial whether plesiosaurs flew underwater, rowed, or used a mixture of the two modes of locomotion. The long bones of Tetrapoda are functionally loaded by torsion, bending, compression, and tension during locomotion. Superposition of load cases shows that the bones are loaded mainly by compressive stresses. Therefore, it is possible to use finite element structure analysis (FESA) as a test environment for loading hypotheses. These include muscle reconstructions and muscle lines of action (LOA) when the goal is to obtain a homogeneous compressive stress distribution and to minimize bending in the model. Myological reconstruction revealed a muscle-powered flipper twisting mechanism. The flippers of plesiosaurs were twisted along the flipper length axis by extensors and flexors that originated from the humerus and femur as well as further distal locations.Cryptoclidus eurymerus (Middle Jurassic Oxford Clay Formation from Britain) were analyzed using FE methods based on the concept of optimization of loading by compression. After limb muscle reconstructions including the flipper twisting muscles, LOA were derived for all humerus and femur muscles of Cryptoclidus by stretching cords along casts of the fore- and hindflippers of the mounted skeleton. LOA and muscle attachments were added to meshed volumetric models of the humerus and femur derived from micro-CT scans. Muscle forces were approximated by stochastic iteration and the compressive stress distribution for the two load cases, “downstroke” and “upstroke”, for each bone were calculated by aiming at a homogeneous compressive stress distribution.To investigate locomotion in plesiosaurs, the humerus and femur of a mounted skeleton of Humeral and femoral depressors and retractors, which drive underwater flight rather than rowing, were found to exert higher muscle forces than the elevators and protractors. Furthermore, extensors and flexors exert high muscle forces compared to Cheloniidae. This confirms a convergently evolved myological mechanism of flipper twisting in plesiosaurs and complements hydrodynamic studies that showed flipper twisting is critical for efficient plesiosaur underwater flight. Plesiosauria are secondarily aquatic tetrapods that lived in the sea from the Late Triassic  until thi.e., Cheloniidae + Dermochelyidae)). Unlike plesiosaurs, only the foreflippers of penguins, sea turtles, whales and dolphins (Cetacea), and sea lions (Otariinae) are hydrofoil-like flippers  , and forflippers . The hinflippers  which arflippers . The hinCryptoclidus eurymerus, a Middle Jurassic cryptoclidid known from many specimens from the lower Oxford Clay Formation of England  shows these features well. The humeral and femoral heads of plesiosaurs were probably covered by a thick and vascularized cartilaginous cap  gastrocnemius)   or wallacnemius) . In contba livia  or aboutrhynchos . In generhynchos .As measurements with strain gauges show, the long bones of Tetrapoda are functionally loaded by torsion, compression, and bending in an intermittent fashion. Most often they are loaded by bending in alternate directions or mainly by compression and subordinately by tension. High torsional loads act on the long bones of terrestrial tetrapods .To represent such changing loading conditions, a series of load cases can be analyzed using finite element structural analysis FESA;  and thenvia tension chords. Tension chords are either muscles and tendons (active tension chords) or ligaments (passive tension chords), and they act in pairs of agonists and antagonists  exhibited at the Goldfuß Museum, Section of Paleontology, Institute of Geosciences, University of Bonn (IGPB), on which the muscle reconstructions of LOA represent the direct connection in a straight line between the origin and attachment of a muscle . HoweverDermochelys coriacea and its associated osteological correlates    and the e.g., m. subcoracoscapularis, m. coracobrachialis brevis, m. pectoralis). In the case of the m. latissimus dorsi, for example, a position between the most cranial and the most caudal origin was chosen. This was done to represent the parts that are best supported by the EPB , but also to cover the less well supported part   were taken from al part) .e.g., m. pectoralis, m. latissimus dorsi, m. puboischiofemoralis internus, and m. puboischiofemoralis externus. Muscles with two or more heads , m. flexor tibialis internus, m. flexor tibialis externus, m. puboischiofemoralis internus, and m. puboischiofemoralis externus) were implemented as two (or more) cords. The resulting vectors of the different subsections of these muscles, which have a large area of origin, were included in the FE models. Agonistic and antagonistic muscles attaching to, originating from, or spanning the humerus   and 7B, We chose not to refer to the neutral flipper position as the resting position of the flipper because it is probably impossible to determine the resting position of the flipper for an extinct species. Furthermore, in the muscle physiology literature, the term “resting length” usually refers to individual sarcomeres  or fascii.e., maximum deflection on downstroke, neutral position, and maximum deflection on upstroke, were measured in cm using a tape measure as the distance between the respective terminal strips. The changes in length between the maximum deflection on the downstroke and the neutral position and between the maximum deflection on the upstroke and the neutral position were recalculated as percentages, with the resting length set to 100%. Then, muscle stretching, muscle contraction, and the difference between the two, the total change in muscle length, were calculated in % . Hence, a different area of origin  was tested, which yielded physiologically plausible results, which were then measured for all three positions.All three positions of the fore- and hindflippers were fixed in sequence with black nylon cords suspending the flippers from the wooden frame. For each of the three positions, the length of each muscle was fixed using the terminal strips by tightening the small screws in the terminal strips on the muscle cords. Then, each muscle cord was removed from the model by unhooking it from the screw eye pins, and all three muscle lengths, ted in % . Bar grated in %  and 8B. z-direction for the humerus and femur.FESA requires a 3D model of the bones to be investigated. Hence, the right humerus and left femur were removed from the mounted skeleton IGPB R 324 and scanned with the micro-CT scanner at IGPB. The scanner is an industrial high-resolution computed tomography scanner . The scans were processed using the dedicated datos|x software and the VGStudio MAX (Volume Graphics) program to obtain image stacks in the i.e., cortical vs. spongy, because this would have precluded implementation of torsion in the FESA. The reason is that the much stiffer cortex would take up all the stress peaks.The two image stacks were loaded into Simpleware ScanIP 5.1  for furtThe 3D model was then imported into ANSYS 16.0  as the .stl file. The dimensions of the humerus and femur from IGPB R 324 were used to scale the respective volumetric model to the original size. Subsequently, the proximal articular cartilage or articular surface between the glenoid and acetabulum was modeled as a volume. Bonded contacts between the artificial cartilage structures were implemented in the analysis. The models were constrained by fixed boundaries on the upper surface of the cartilage volume. Similar to other established finite element models , the matNext, the LOA and muscle insertions had to be implemented in the FE model. We did this by taking photographs of the analog model in anterior, posterior, dorsal and ventral views and in these traced the LOA and their angles of attachment on the humerus  and femuForce transmission to the distal articular surfaces of the humerus and femur due to muscle activity was initially applied in one point. This resulted in artificially high, localized stress peaks. Therefore, the force application was divided into several application points distributed over the distal articular surfaces to perform a more realistic simulation of load application over one surface.The stress distribution was calculated for both bones –10N. TheTwo load cases, downstroke and upstroke, were selected for calculation to reflect the constantly changing load on the humerus and femur during the flipper beat cycle –10J. TheFor both load cases, a position was chosen in which the humerus was held horizontally at the level of the glenoid and pointed laterally and slightly forward, as in the analog model. Similarly, the femur was positioned horizontally at the level of the acetabulum with the flipper tip pointing mainly laterally, but also angled slightly posteriorly. On the downstroke, the humerus and femur were also rotated anteriorly downward around their long axis by approximately 19°  to modelFor the implementation of load cases, it is crucial to identify and consider which muscles act as agonists and antagonists . The shoTo identify agonists and antagonists, opposing muscle functions  and 3 arAs detailed above, humeral muscles reconstructed based on comparative anatomy  were tesvia a common tendon or on the posteroproximal part of the radius and anteroproximal part of the ulna. In addition, the m. triceps brachii was suggested to originate anterodorsally from the bony ridge surrounding the glenoid on the scapula and from the coracoid immediately posterior to the glenoid and suggested to insert into the posterodorsal ulna .M. coracobrachialis brevis, m. coracobrachialis longus, m. biceps brachii, the large posterior portions of m. pectoralis, m. subcoracoscapularis, m. supracoracoideus and m. latissimus dorsi are humeral retractors. They are opposing the humeral protractors, namely the m. deltoideus clavicularis, m. deltoideus scapularis, and the small anterior portions of the m. supracoracoideus, m. subcoracoscapularis, and m. latissimus dorsi. The m. deltoideus scapularis, the m. subcoracoscapularis, the m. latissimus dorsi, and possibly to a very small extent the m. triceps brachii, the m. scapulohumeralis anterior, and the m. scapulohumeralis posterior cause elevation of the humerus. The humeral depressors m. supracoracoideus, m. coracobrachialis brevis, m. coracobrachialis longus, m. deltoideus clavicularis, m. biceps brachii, and m. pectoralis act antagonistically to the humeral elevators. The m. triceps brachii, the smaller anterior portion of the m. subcoracoscapularis, the large posterior portion of the m. pectoralis, the m. biceps brachii, the m. scapulohumeralis anterior, the m. scapulohumeralis posterior, and the m. deltoideus clavicularis contribute to the downward rotation of the foreflipper. Functionally antagonistic are m. deltoideus scapularis, m. coracobrachialis brevis, m. coracobrachialis longus, the larger posterior part of m. subcoracoscapularis and m. latissimus dorsi .Further subdivisions into agonistic and antagonistic muscles are possible: The small anterior portion of the m. latissimus dorsi (providing weak elevation and protraction) is opposing in function to the large posterior portion of the m. pectoralis (providing strong retraction and depression). The small anterior m. pectoralis portion (providing protraction and depression) and the large posterior m. latissimus dorsi portion (providing strong elevation and retraction) act as agonist and antagonist. The m. subcoracoscapularis and m. deltoideus scapularis, which elevate and retract the humerus, find their functional antagonists in the m. coracobrachialis brevis, m. coracobrachialis longus, m. biceps brachii, and the posterior portion of the m. supracoracoideus, which depress and retract the humerus. The large posterior portion of the m. subcoracoscapularis acts as a humeral elevator and retractor, and the anterior portion of the m. supracoracoideus and m. deltoideus clavicularis act opposite to them as humeral depressors and protractors .Muscles originating from the humerus aid in flipper twisting : the m. The elevators of the hindflipper are m. puboischiofemoralis internus, m. iliotibialis, m. iliofemoralis, m. iliofibularis, m. caudofemoralis brevis, m. caudofemoralis longus, m. flexor tibialis externus (iliac portion), and m. flexor tibialis internus . M. puboischiofemoralis externus, m. adductor femoris, m. ischiotrochantericus, m. puboischiotibialis, m. flexor tibialis externus  and m. flexor tibialis internus  act as femoral depressors. Protractors are the m. puboischiofemoralis externus (pubic portion), m. puboischiofemoralis internus , m. ambiens, and m. pubotibialis. The retractors of the hindflipper are m. puboischiofemoralis externus , m. puboischiofemoralis internus , m. adductor femoris, m. ischiotrochantericus, m. iliofemoralis, m. iliotibialis, m. caudofemoralis brevis, m. caudofemoralis longus, m. flexor tibialis externus, and m. flexor tibialis internus. The downward rotation of the flipper leading edge during the downstroke is controlled by the agonists m. puboischiofemoralis internus (pubic portion), m. puboischiofemoralis externus , m. caudofemoralis brevis, m. caudofemoralis longus, m. ambiens (when the femur is elevated), m. ischiotrochantericus, m. iliofibularis (as long as the fibula is below the origin of these muscles), m. puboischiotibialis, m. pubotibialis (when the femur is elevated), m. flexor tibialis internus and m. flexor tibialis externus. The agonists are opposed by the antagonistically acting m. iliotibialis, m. ambiens (when the femur is depressed), m. pubotibialis (when the femur is depressed), m. iliofemoralis, m. puboischiofemoralis internus  and m. puboischiofemoralis externus (large pubic portion) .In addition, the muscles can be assigned to the following subgroups: The m. puboischiofemoralis internus, which originates from the dorsal pubis and vertebral column, is opposed by muscles originating from the ventral ischium , m. adductor femoris, and the ischial portions of m. flexor tibialis internus and m. flexor tibialis externus). The pubic portion of m. puboischiofemoralis externus, m. ambiens and m. pubotibialis, which originate from the ventral region of the pubic bone, have muscles originating from the ilium and posterior vertebral column as antagonists ). Musculus ambiens and m. pubotibialis have m. iliofibularis as antagonist. M. femorotibialis and m. extensor digitorum longus mostly originate dorsal to the femur. M. gastrocnemius externus, m. gastrocnemius internus and m. flexor digitorum longus arise ventrally from the femur. M. gastrocnemius externus and m. gastrocnemius internus as well as m. flexor digitorum longus (flexion of the toes) appear to be opposite to m. extensor digitorum longus (extension of the toes) and m. femorotibialis .Compressive stress distribution and muscle forces were calculated for the downstroke and upstroke load cases of the humerus FE model. For the initial FESA runs, the dorsal tuberosity of the humerus was left unloaded. Augmenting the FESA to include wrapping of the m. latissimus dorsi and m. subcoracoscapularis around the dorsal tuberosity, which resulted from LOA observations, helped to apply compressive stress to this process. In addition, extensor and flexor muscles wrapping around the anterior and posterior distal processes (ectepicondylar and entepicondylar processes) of the humerus allowed their loading by compressive stress. This is because muscles that wrap around a bone exert compressive stress on it. In contrast, muscles that do not wrap around the bone exert only local tensile stress on the bone.In the FESA results for the humerus  and 9L, On the distal articular surface of the humerus, high stress peaks occur in a very confined manner  and 9K. i.e., m. latissimus dorsi and m. subcoracoscapularis. Nevertheless, both, m. subcoracoscapularis  and m. latissimus dorsi , develop high forces to maintain the upstroke together. In general, it appears that greater muscle forces are generated by the retractors and depressors of the humerus than by its elevators and protractors. In addition, extensors and flexors sometimes develop extremely high muscle forces, with the m. flexor carpi radialis generating 8,460 N on the downstroke and the m. extensor digitorum communis generating 6,000 N on the upstroke  among the muscles spanning the glenoid. Surprisingly, on the upstroke, the m. pectoralis still develops a higher force  than either of the two main humeral elevators, upstroke .There is a wide range of values for the Young’s modulus of bone in the literature, and the value of 12,000 MPa chosen in this study is a relatively low estimate. We justify our choice of this low value because the flipper model is made of a homogeneous material. A higher value would not change the stress distribution or the stress level but would affect the strain: a higher Young’s modulus is associated with lower strains in linear elastic materials.The compressive stress distribution and muscle forces were calculated for the down- and upstroke load cases of the femur FE model. In the initial femur FESA runs, we were unable to apply compressive stresses to the dorsal trochanter and distal epiphyses of the femur because the muscles would simply pull away from their origin. Thus, only localized tensile stresses were observed in the FESAs. We then introduced muscle wrappings as in the humerus, with the m. iliofemoralis and m. puboischiofemoralis internus wrapped around the dorsal trochanter and the extensors and flexors wrapped around the distally greatly expanded epicondyles of the femur. This resulted in these structures being loaded by compressive stress.Color coding of compressive stress distribution in the plesiosaur femur  and 10L The muscle forces of the many two-joint muscles  in the hindflipper cannot be determined, because they influence the femur only indirectly by contributing to the counterforce. On the downstroke, the m. puboischiofemoralis externus generates the highest muscle force . On the upstroke, the m. puboischiofemoralis internus generates up to 7,611 N. The forces of the extensor and flexor muscles are much lower in the femur than in the humerus. The gastrocnemius muscle, a flexor muscle, develops a total force of up to 1,176 N .Muscles originating dorsally from the glenoid and acetabulum extend on the downstroke and contract on the upstroke. Muscles originating ventrally from the glenoid and acetabulum contract when the humerus and femur are depressed on the downstroke and lengthen on the upstroke when the humerus and femur are elevated.i.e. the length changes were not measurable with the technique used here, i.e. they are smaller than 1.7 cm . The m. coracobrachialis brevis (posterior part) shows very small muscle shortening (3.88%). Otherwise, the changes in total muscle length cover the entire physiological spectrum, from about 9% in the posterior part of the m. subcoracoscapularis to 37% in the anterior part of the m. latissimus dorsi. The only muscle that stands out is the m. deltoideus scapularis with a total length change of over 70%. This is clearly not physiological. Therefore, a screw eye pin was screwed into a different hypothetical region of origin of the m. deltoideus scapularis. This different origin was located at the ventral to ventrolateral scapula anterior to the glenoid. The total muscle length change was again measured in all three flipper positions and was now within the measurement error of the analog model and well within physiological limits  ) show no length change, l limits .Total muscle length changes for the hindflipper range from 0% to 35.8%. We detected no length changes for the m. caudofemoralis brevis (ilium portion) and the m. pubotibialis. In addition, the m. caudofemoralis brevis, which originates from the vertebral column, shows little change in total muscle length (5.2%), while the muscle with the greatest change (35.82%) in total length is the part of the m. puboischiofemoralis internus that originates from the vertebral column .e.g., m. puboischiofemoralis externus (23.55%)), for example, exhibited similar shortening to a muscle originating from the anterodorsal pubis ).Considering the agonistic and antagonistic muscles, the total length changes of m. pectoralis and m. latissimus dorsi, the two muscles that mainly drive the downstroke and upstroke of the foreflipper, are quite similar: the anterior part of m. latissimus dorsi (36.96%) and the posterior part of m. pectoralis (35.65%), and the posterior part of m. latissimus dorsi (21.92%) and the anterior part of m. pectoralis (18.75%). We expected that the overall changes in muscle lengths of agonists and antagonists would be similar because of their opposite functions. Instead, we found that the muscles exhibiting comparable total length changes were more likely to be determined by their geometric arrangement relative to the glenoid or acetabulum. That is, a muscle originating from the posteroventral ischium  .The muscle reconstructions on which this study is based were obtained by evaluating comparative anatomical data (the EPB) . These bOne muscle, the m. scapulohumeralis anterior, was added here to the reconstructions of M. scapulohumeralis anterior occurs exclusively in lepidosaurs and not in turtles or crocodylians . In lepiWe reconstructed m. scapulohumeralis anterior in agreement with i.e., a small change, would better account for the often non-parallel and rather complex architecture of the deltoid muscle  could free or largely free the ilium from locomotor muscles, allowing for its reduction in size.Total length changes were calculated for glenoid and acetabular muscles and tested to see if they were within physiological limits. If it had originated from the lateral scapula, the total length change of the m. deltoideus scapularis is not physiological and would not allow the muscle to produce much force see  Table 1. This mae.g., m. deltoideus clavicularis, both m. triceps brachii heads, m. coracobrachialis brevis (posterior part); m. caudofemoralis brevis (ilium), m. pubotibialis) have similar length changes for elevating and depressing the humerus and femur. If protraction and retraction were also considered, as well as rotation of the humerus about the long axis, the muscle length changes would give different results for all these muscles. It is possible that these nearly isometrically contracting muscles had a complex muscle architecture . Another possibility is that the muscles that do not show length changes were reduced in plesiosaurs. Some clues might be provided by the EPB: The m. triceps brachii is greatly reduced or completely reduced in Chelonioidea, depending on the species. The m. coracobrachialis brevis is significantly reduced in Testudines in general  compared to the m. extensor digitorum communis . M. flexor carpi radialis and m. flexor digitorum longus are topologically arranged similarly to m. extensor carpi ulnaris and m. extensor digitorum communis, but on the ventral side of the foreflipper. In contrast, the m. flexor carpi radialis and m. flexor digitorum longus develop much lower muscle forces  . HoweverCryptoclidus (as opposed to many other plesiosaurs), and it is possible that the hindflipper contributes less to propulsion than the foreflipper, as suggested by When comparing the muscle forces of the extensor and flexor muscles of the plesiosaur foreflipper and hindflipper, it is noticeable that those acting on the femur  are geneCryptoclidus eurymerus and in Cheloniidae: The m. pectoralis develops the highest force of all muscles that attach proximally to the humerus  in the shoulder girdle were large and occupied a lot of space. Therefore, it is problematic that the extensor and flexor muscles were inferred to generate similarly large forces because their potential areas of origin are much smaller than those of the glenoid-spanning muscles. There may be several solutions to this paradox. For example, the m. flexor digitorum longus in Sauropsida has a second head originating from the carpus , so it mAnother possibility, how large muscle forces in distal muscles may have been attained, can be observed in dolphins. Dolphins have a relatively well ossified flipper skeleton, although essentially no individual muscles are recognizable. They have only layers of parallel, fibrous connective tissue covering the flipper bones see , p. 1128Cryptoclidus based on the mounted skeleton IGPB R 324. These models are consistent with underwater flight in plesiosaurs based on comparative anatomical and muscle physiological data in accordance with mechanical principles.The highly aquatic-adapted locomotory system of plesiosaurs changed little throughout the 135 million years of plesiosaur evolution. The question of whether plesiosaurs rowed, flew underwater, or used a combination of both has not yet been fully answered. Here we present analog muscle reconstructions and a computer model of the plesiosaur i.e., the dorsal tuberosity of the plesiosaur humerus and the dorsal trochanter of the plesiosaur femur, as well as their epicondyles, proved to be necessary to load the bony structures. Measurements in an analog model of the total length changes of all muscles attaching to, originating from, and spanning the humerus and femur of a plesiosaur revealed that an origin of the m. deltoideus scapularis at the lateral scapula is unphysiological. This suggests a reduction of this muscle from this part of its site of origin, which is well documented in Sauropsida (We show that it is possible to test muscle reconstructions and associated lines of action with FESA by targeting a homogeneous compressive stress distribution in the humerus and femur. The muscle reconstructions of uropsida . Muscle 10.7717/peerj.13342/supp-1Supplemental Information 1Click here for additional data file.10.7717/peerj.13342/supp-2Supplemental Information 2Click here for additional data file.


Escherichia coli (E. coli) drug resistance in large-scale pig farms. In this study, 296 strains of E. coli isolated and identified from large-scale pig farms in Beijing were used as the research objects. In vitro drug sensitivity tests were used to determine the sensitivity to 10 antibiotics of pig-derived E. coli. SPSS logistic regression was employed to analyze the effects of the season, pig type, sampling point (medication type) and sampling location on resistance and multi-drug resistance of E. coli from pigs. The degrees of drug resistance to 10 antibiotics of the 296 strains of pig-derived E. coli were varied, their resistance rates were between 4.05 and 97.64%, and their multi-drug resistance was appalling, with the highest resistance to six antibiotics being 26.35%. The isolated strains were proven more resistant to tetracyclines, penicillin and chloramphenicol, which are commonly used for disease prevention in pig farms, and less resistant to quinolones and aminoglycosides, which are not used in pig farms. The resistance of the isolated strains in spring and summer was generally higher than that in winter. E. coli resistance in piglets, fattening pigs and sows was more serious than that in nursery and sick pigs. The results showed that the season, type of medication and type of pig had an influence on the pig-derived E. coli resistance, among which the type of medication was the most influencing factor.With widespread use of antibiotics in the aquaculture industry, bacterial resistance has recently attracted increasing attention. Continuous emergence of multi-resistant bacteria has greatly threatened human and animal health, as well as the quality and safety of livestock products. To control bacterial resistance, the effect of bacterial resistance needs to be well understood. The purpose of this study was to explore the factors influencing  Antibiotic resistance (AR) affects treatment of bacterial diseases in humans and animals and will become one of the major threats facing the world in the future . The maiEscherichia coli (E. coli) is an intestinal symbiotic bacterium that exists in animals and humans and is susceptible to high selection pressure from antimicrobial agents in contact with the host. Therefore, E. coli is often used as a carrier for monitoring antimicrobial resistance in human or livestock groups  is increasing worldwide, which has attracted extensive attention  of pig-derived E. coli, including the season, pig type, sampling position (medication type) and sampling location. This study explored the main factors affecting AR of E. coli in pigs and provided a scientific basis for rational use of drugs in large-scale pig farms to control growing bacterial drug resistance.To understand the factors important for antimicrobial resistance of E. coli derived from sick pigs, piglets, sows, nursery pigs, and fattening pigs.Large-scale pig farms in Shunyi District of Beijing were selected for a visit. The herds were farrow-to-finish herds with at least 800 sows, generating 12,000 heads every year. Herds were visited three times to collect samples for antimicrobial resistance profiling of  swabs from E. coli by BLAST . All isolates confirmed as E. coli were tested for antibiotic resistance.After collection, the swab samples were transported to the laboratory where they were processed within a few hours after arrival  standards were followed for inoculum standardization, incubation conditions and internal quality control organisms  and OR ≠ 1.Microsoft Excel was used to include all data, and all the data were classified and a library was constructed using Statistical Product and Service Solutions  software. Single-factor binary logistic regression was utilized to analyze the effects of four factors, including the season, type of pig, sampling position and sampling location, on resistance of E. coli was analyzed, and specific assignment instructions were listed in P-value of the OR value helped screen out possible associations with MDR of E. coli. Significant factors that affect MDR of E. coli were analyzed by adding the selected influencing factors to multi-factor multiple logistic regression analysis based on the magnitude of the OR value with P < 0.05 as the significant difference and OR ≠ 1, and then the main way that could affect MDR of E. coli was analyzed.By single-factor multiple logistic regression, the influence of four factors on MDR of E. coli were tested for their resistance to 10 antibiotics by drug susceptibility. From A total of 296 strains identified as E. coli resistance in different seasons was evaluated, and the results were shown in Herein, pig-derived E. coli resistance, binary logistic regression analysis was employed. AR of E. coli isolated from the samples collected in winter was used as a reference and the results were given in E. coli sampled in spring and summer to all the 10 antibiotics except tetracycline. Compared with E. coli sampled in winter, what was sampled in spring and summer showed a significant difference in resistance to florfenicol, and the OR value of florfenicol (2.963) was higher than other antibiotics in spring. Moreover, E. coli resistance to gentamicin and erythromycin in summer was significantly different from what was in winter, and the OR values were 3.411 and 1.894, respectively.In order to better understand the influence of seasons on E. coli isolated from different types of pigs  was evaluated, and the results were shown in Resistance of E. coli, binary logistic regression analysis was exploited in this study. AR to 10 antibiotics of E. coli isolated from piglets was used as a reference, and all the results were shown in E. coli in nursery pigs and sick pigs was relatively serious. Compared with piglets, significant differences existed in resistance to florfenicol of E. coli from nursery pigs, fattening pigs and sick pigs, and the resistance to florfenicol was very serious in nursery pigs (OR = 12.294). Compared with piglets, resistance to chloramphenicol of E. coli from nursery pigs, fattening pigs and sick pigs was also significantly different, and resistance of E. coli in nursery pigs was more serious than that in fattening pigs and sick pigs (OR = 6). Similarly, compared with piglets, resistance to erythromycin of E. coli from nursery pigs was significantly different (OR = 7). Compared with piglets, there were considerable differences in gentamicin resistance among fattening pigs, sows and sick pigs. By comparing the OR values, it could be found that resistance of piglets to gentamicin was more serious. The results of resistance to florfenicol, chloramphenicol, erythromycin, and gentamicin of E. coli isolated from different pig types obtained by binary logistic regression analysis were consistent with those obtained by drug resistance analysis. In short, a significant correlation was presented between pig types and E. coli resistance.To understand the influence of pig types on resistance of E. coli on antibiotic sensitivity was analyzed according to different sampling sites, and the results were shown in The influence of E. coli, binary logistic regression analysis was utilized. Resistance to 10 antibiotics of E. coli isolated from nasal swabs was used as a reference and the results were listed in E. coli from anal and nasal swabs, and the OR values were 0.589, showing that E. coli from nasal swabs was more resistant to florfenicol.To understand the influence of sampling positions on resistance of E. coli from pigs was statistically analyzed according to different sampling locations, and the results were shown in The antibiotic sensitivity of E. coli resistance. Chloramphenicol, banned for veterinary use, possessed a high drug resistance rate for E. coli from pigs, similar to florfenicol. The possible reason is that both drugs belong to the class of chloramphenicol and have been affected by the spread of resistance genes.The tested bacteria isolated from the southern area had higher resistance to florfenicol and chloramphenicol, reaching the resistance rates of 80.21 and 62.5%, respectively, compared with those from the northern area, with their respective resistance rates of 49.04 and 39.42%. The differences in drug use in the northern and southern areas may be the reason for disparate E. coli, binary logistic regression analysis was utilized. The resistance to 10 antibiotics of E. coli isolated from the northern area was used as a reference and the results were listed in E. coli sampled from the southern area to all the 10 antibiotics except tetracycline. Moreover, resistance to florfenicol, chloramphenicol and erythromycin of E. coli from the southern area was significantly different from what was from the northern area, and the OR values were 4.035, 2.561, and 1.82, respectively.To understand the influence of sampling locations on resistance of E. coli resistance. There was a significant correlation between AR of E. coli and the sampling season, pig type and sampling location. The three factors were introduced into the logistic regression equation for multivariate logistic regression analysis. It could be seen from E. coli resistance to all antibiotics except tetracycline in the southern area was higher than that in the northern area.Single-factor binary logistic regression analysis was applied to analyze the relationship between the four factors and pig-derived From E. coli from pigs. The rates of resistance to all antibiotics except gentamicin in nursery pigs were higher than that in piglets , revealing that E. coli in winter tended to be more multi-drug resistant than that in spring. Meanwhile, MDR of E. coli in summer was 0.629 times higher than that in winter. Although there was no significant difference, E. coli tended to be more extremely resistant in summer than in winter. In spring and summer, the prevalence of E. coli resistance was more serious.According to E. coli MDR results were statistically analyzed according to different pig types, as shown in The E. coli from nursery pigs were 0.229 times that of piglets , indicating that E. coli from nursery pigs was more likely to obtain extreme drug resistance than piglets. It also showed that the distribution of E. coli resistance in piglets and sows was similar. The comparison of the OR values showed that there was little difference in MDR between piglets and sows (OR = 1.013). From the analysis of different pig types, the extreme drug resistance of E. coli in nursery pigs was more serious, and there was a possibility of vertical transmission from sows to piglets.According to E. coli MDR was statistically analyzed according to different sampling sites of E. coli from pigs, and the results were displayed in E. coli from nasal swabs and anal swabs was mainly concentrated in 5–6 antibiotics and the results were listed in E. coli from nasal swabs was mostly concentrated in the MDR category, and extreme resistance of E. coli from anal swabs was more serious. Meanwhile, there was no significant difference between the two. Therefore, the sampling position was not used as a dependent variable for multi-factor multinomial logistic analysis.E. coli from pigs was analyzed according to different sampling locations, and the results were shown in E. coli was more serious in the southern area.MDR of E. coli in the northern area were 4.851 times higher  than those in the southern area. Resistance of E. coli in the southern area was more prone to extreme drug resistance than that in the northern area. Analysis from different sampling locations demonstrated that the prevalence of MDR and resistance to 10 antibiotics of E. coli in the southern area was higher than those in the northern area, indicating that the sampling location was an important factor affecting E. coli drug resistance.According to E. coli from pigs. Among them, the sampling season, pig type and sampling location were significantly associated with MDR of pig-derived E. coli. The three factors were introduced into logistic regression equation to carry out multi-factor multiple logistic regression analysis. According to E. coli in the northern area was 4.113 times that in the southern area , which indicated that E. coli in the southern area was more likely to produce extreme drug resistance than that in the northern area. After logistic regression analysis of the three factors at the same time, only the sampling location was significant, revealing that it was the main factor affecting MDR of E. coli from pigs. The biggest difference between the two sampling locations was caused by the type of drug used. The use of antibiotics was an important factor affecting extreme AR of E. coli from pigs.Single-element multiple logistic regression was used to analyze the relationship between the four factors and MDR of E. coli isolated from pig farms in spring was detected by paper agar amplification. There was only one strain (1.18%) of E. coli that can produce ESBLs, and the results were given in The expression of extended spectrum β-lactamases (ESBLs) in 85 strains of E. coli based on types of drugs, pig types, seasons and sampling locations, it is possible to find out the risk factors that trigger bacterial resistance in the pig breeding process and to analyze the factors affecting bacterial resistance, which provides a theoretical basis for mitigating the transmission of drug resistance of E. coli.By exploring the differences in resistance of pig-derived E. coli revealed that E. coli is especially sensitive to quinolones and aminoglycoside drugs, highly resistant to tetracyclines and penicillin, and relatively resistant to sulfonamides and chloramphenicol. The rate of E. coli resistance to erythromycin is low, mainly concentrated between susceptibility and resistance, indicating that E. coli is potentially resistant to erythromycin. This warns farmers that use of macrolide drugs should be controlled to reduce the risk of E. coli resistance to macrolide drugs. Pig-derived E. coli is mainly resistant to 5–6 kinds of antibiotics, and the ratio of resistance to more than 3 kinds of drugs is 85.47%, indicating that resistance of E. coli in farms is relatively serious. Similar E. coli resistance results have been found in the study that tested pig farms in France, Italy, Denmark and Sweden , suggesting that pig-derived E. coli in global pig farms is highly resistant to tetracyclines, penicillin, and sulfonamides, which may be related to long-term, widespread use of these three types of drugs in the pig-feeding industry.Comprehensive analysis of AR and MDR distribution of pig-derived d Sweden . A studyThailand , which iE. coli clinical isolates in Oceania, Africa, America, Europe, and Asia, which were 0.32, 2.27, 5.19, 25.49, and 66.72, respectively. Their research included 79% of the works published between 2014 and 2020 (E. coli (MCRPEC) isolates from 774 stool samples collected from 5,159 healthy individuals in 30 provinces and municipalities in 2016, with MCRPEC prevalence ranging from 3.7 to 32.7% (average: 15.0%)—substantially higher than previously reported  genes, emerged as a major threat to the control of infections caused by Gram-negative bacteria. and 2020 . Shen etreported .As human aplastic anemia and certain reproductive/hepatotoxic issues in animals could be induced by chloramphenicol, its use in food, feed, medicine and aquaculture production has been banned for at least 20 years in different parts of the world . Since 2E. coli resistance than piglets, fattening pigs and sows, and the epidemic situation of extreme drug resistance of E. coli was more serious in nursery pigs. SPSS logistic regression equation was used to analyze the effects of pig types on AR and MDR of pig-derived E. coli. It was found that E. coli resistance for piglets and nursery pigs in pig farms was more serious, which was similar to the situation in pig farms in Liaoning and other provinces, indicating that most pig farms were facing high E. coli AR in piglets and nursery pigs across the country. This may be ascribed to younger ages of piglets and nursery pigs, underdevelopment of their bodies, low immunity and susceptibility to diseases. In the treatment process, resistance of E. coli is further enhanced due to the influence of selective pressure. The spread of infectious diseases in pig farms is affected by many factors such as the feeding mode and disease control, herd size, biosecurity and sanitation level  and for therapeutic purposes  in the Netherlands revealed a relationship between antimicrobial use and antimicrobial resistance  of E. coli resistant to all tested antibiotics, defining its relatively high resistance.A total of 85 isolated pig-derived E. coli isolated from pigs were analyzed and it was found that there were significant differences in AR and MDR of E. coli from pigs. Among these factors, drug types significantly affected AR and MDR of E. coli isolated from pigs. In conclusion, the seasonal change, selective drug use and type of pig have impacts on AR of E. coli from pigs according to SPSS logistic regression analysis. The experimental comparison results implied that the medication type was the main factor affecting AR of E. coli.In this paper, the effects of seasons, pig types, sampling positions (medication type) and sampling locations on drug resistance and MDR of The original contributions presented in the study are included in the article/SY conceived and designed the experiments. QL and YC provided the detection samples, statistical and data analysis in this research. RZ and JW contributed to the reagents and materials. XL prepared the draft of this manuscript. RL and YW collected the samples. AC revised the article and checked the figures. All authors have read and agreed to the published version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Nowadays, with numerous sensors placed everywhere around us, we can obtain signals collected from a variety of environment-based sensors, including the ones placed on the ground, cased in the air or water, etc. These sensors can produce various signals, such as audio, image, video, infrared data, and so on. The obtained signals are usually processed using dedicated software, utilizing the output of these sensors. The construction of optimized sensors, the development of the theory that can he applied for signal representation and analysis, as well as the design of software for the processing of the obtained data represent broadly explored research topics. Therefore, recent advances in these areas are of interest for many researchers, and for the users of sensing techniques.The Special Issue entitled “Analytics and Applications of Audio and Image Sensing Techniques” is focused on original research involving the use of various audio and image sensing devices, both simultaneously and separately. It collected a diverse set of papers that span a wide range of analyses and possible applications.Digital signal processing;Audio signal analysis;Image analysis;Pattern recognition.Topics for the Special Issue included the following:Altogether, 17 papers have been included in this Special Issue.The papers presented in this Special Issue cover a broad range of topics, from the theory of signal analysis, to practical applications of audio and image sensing techniques. Audio signals discussed in these papers cover both speech and music, including measurements of airflow in organ pipes. Image and video data analyzed in the presented papers include microscope images and infrared images, as well as face images. All papers are briefly presented below.The paper  by A. StSix contributions in this Special Issue are dedicated to audio signal, including both music and speech.The paper  by M. BlP. Wrzeciono in his paper  presentsThe sound of organ pipes is also addressed in the paper  by D. WęP. Odya, J. Kotus, A. Kurowski, and B. Kostek in  analyzedIn the next paper , A. KuroK. Szklanny and J. Lachowicz in  describeFive papers in this Special Issue deal with image processing, including face images, microscopic images, and infrared images.A. E. Mahdaoui, A. Ouahabi, and M. S. Moulay in  propose M. Geremek and K. Szklanny in  investigIn , E. KubeThe next paper , writtenL. C. M. Dafico, E. Barreira, R. M. S. F. Almeida, and H. Carasek in  use infrFive papers in this Special Issue present techniques related to the processing and usage of video signal and audiovisual data.G. Canet Tarrés and M. Pardàs in  propose In paper , F. VossK. Hirayama, S. Chen, S. Saiki, and M. Nakamura in  describeThe study presented in  by J. QuK. Szklanny, M. Wichrowski, and A. Wieczorkowska in  present Sensors will find many of them interesting and inspiring.The papers collected in this Special Issue present a wide range of topics in audio, image, and video domains. I do hope the readership of Sensors, for their hard work in successful preparation of this Special Issue.I would like to thank all the authors who submitted papers to this Special Issue, the reviewers for their efforts in improving the papers, and the editors of


Chlamydia psittaci, is widespread throughout the world. In humans, C. psittaci infection may lead to severe conditions and complications, including sepsis and multiple organ failure. We report a cluster of cases caused by C. psittaci in Zhejiang Province, 2021, which led to one death and three cases of hospitalization.Psittacosis, caused by The cases were confirmed by nest-PCR, RT-PCR, and mNGS.The four cases were related and the sequences obtained from the samples were closely correlated with those from Taiwan.C. psittaci.This study is the first to report on the case of death from psittacosis in Zhejiang Province, and our results help to assess the disease and recommend effective measures to prevent further spread of  Chlamydia psittaci infection. C. psittaci is a Gram-negative pathogen that infects humans throughout the world   .C. psittaci, drawing attention to psittacosis and strengthening disease surveillance.In this study, we report a cluster of psittacosis cases in Lishui, Zhejiang Province, China, in 2021, which caused one death and three cases of hospitalization. Among the four patients with confirmed psittacosis, three patients belonged to the same family and all four cases were connected. To the best of our knowledge, this study is the first to report on the case of death from psittacosis in Zhejiang Province in recent years, and can help in further research on transmission of C. psittaci pneumonia was confirmed by the presence of C. psittaci sequence reads in his bronchoalveolar lavage fluid and blood samples by mNGS. The other three patients , all from Longquan and with C. psittaci infection, were related to Patient 1 and belong to the same family. Patient 2 (65 years old) once bought ducks from Patient 1 and was hospitalized on August 28, 2021, due to severe fever and chills. The condition of Patient 2 deteriorated quickly, and the patient died on September 13, 2021. His wife, Patient 3, developed chills, fatigue, and nausea on August 28, 2021, and was hospitalized for treatment on August 30, 2021. Their son, Patient 4, who attended and stayed with Patients 2 and 3 in the hospital, was hospitalized on September 5, 2021, due to severe fever and chills. The time of disease onset is shown in Patient 1 was from Qingyun County and was hospitalized on August 21, 2021, with fever and chills. Five specimens, including one bronchoalveolar lavage and four serum samples from the four hospitalized patients, were collected from the Central Hospital of Lishui, Zhejiang Province. A total of 74 environmental samples (30 from Patient 1 and 44 from the village of the other three patients), including throat swabs, anal swabs, dung, and drinking water of ducks, chicks, or geese were collected from the patients’ homes.C. psittaci locus tag 16S rRNA and nested PCR ). The results of nested PCR were confirmed by sequencing at Sangon Biotech Company  and BLAST (http://blast.ncbi.nlm.nih.gov/).The total DNA of the samples was extracted using a commercial kit , according to the manufacturer’s instructions. The DNA was analyzed by real-time PCR targeting the . psittaci-positive samples by sequencing the nested PCR products. The primers and probes used are displayed in A total of 11 sequences (ZJLS001-011) were successfully obtained from 15 CPatient 1 was from Qingyun County, while the other three patients were from Longquan, the neighboring county of Patient 1. As shown in C. psittaci accounted for 96.39% and 77.32% of microbial reads at the genus and species levels, respectively  for mNGS. The mNGS results revealed that 8905 and 7144 sequence reads corresponding to C. psittaci in RT-PCR. In addition, two environmental samples (dung and drinking water of goose) from Patient 1 and six environmental samples  from the other three patients (belonging to the same family) were positive for C. psittaci  for the publication of any potentially identifiable images or data included in this article.Methodology: ZW. Formal analysis: GS. Writing—original draft preparation: LW. Writing—review and editing: WY, XC. Supervision: ZY. Project administration: YZ, BW. All authors have read and agreed to the published version of the manuscript. All authors contributed to the article and approved the submitted version.


The need for information on plant conservation techniques is increasing rapidly in the face of increased extinction risks to plants including habitat loss, unsustainable harvesting, and climate change . A fresh infusion of resources, including expertise and funding, is necessary to build global capacity in the conservation of exceptional species, and increased awareness and education can be one means of achieving this. As more researchers study and develop conservation protocols for these vulnerable species and publish their results, collaboration, resources, and efficiency within the exceptional plant conservation community should increase. We hope that this special issue can contribute to that goal.This special issue grew out of a number of collaborative exceptional plant conservation initiatives, including the collaborative grant approach detailed in Philpott et al.  and the M.P. prepared the first draft of the manuscript. M.P. and V.C.P. provided select article summaries. V.C.P. and E.E.D.C. provided reviewing and editing assistance. All authors approved the final version of the manuscript.