Screening for SARS-CoV-2 and Other Coronaviruses in Urban Pigeons (Columbiformes) from the North of Spain under a 'One Health' Perspective.

Coronaviruses have a major impact on human and animal health. The SARS-CoV-2, a beta coronavirus responsible for the COVID-19 pandemic, is a clear example. It continues circulating and causes human deaths, and its high replication rate results in numerous variants. Coronaviruses adapt to birds and mammals and constitute a serious threat, and new viruses are likely to emerge. Urban pigeons (Columbiformes) are synanthropic birds of great interest from a 'One Health' perspective, due to their interaction with humans and other animals. Aware that they may act as viral reservoirs and contribute to their spread, we aimed to investigate the possible presence of SARS-CoV-2 and other coronaviruses in Columbiformes in the city of Logroño, Spain. Oropharyngeal and cloacal swabs were tested using real-time (N1 and E genes from SARS-CoV-2) and conventional PCR assays (RdRp gene from all coronaviruses). SARS-CoV-2 was not detected. A total of 13.3% of pigeons harbored coronaviruses closely related to Gamma coronavirus (Igacovirus) from Columbiformes in Finland, Poland and China. Monitoring the emergence of a new variant of SARS-CoV-2 capable of infecting Columbiformes should continue. SARS-CoV-2 is still circulating, the viral RNA of this virus has been detected in avian species (Phasianidae and Anatidae), and other coronaviruses are associated with animals that are in close contact with humans. The presence of Gamma coronavirus in urban pigeons must be considered for the risk of surveillance of human infections.

Doxycycline responding illnesses in returning travellers with undifferentiated non-malaria fever: a European multicentre prospective cohort study.

BACKGROUND: Diagnosis of undifferentiated non-malaria fevers (NMF) in returning travellers is a great challenge. Currently, there is no consensus about the use of empirical antibiotics in returning travellers with undifferentiated NMF. Although studies in endemic areas showed that a wide range of pathogens implicated in undifferentiated NMF are treatable with doxycycline, the role of doxycycline in returning travellers with fever still has to be explored. METHODS: Prospective European multicentre cohort study of febrile international travellers (November 2017-November 2019). Immunological and molecular diagnostic techniques for doxycycline responding illnesses (DRI) agents such as Anaplasma phagocytophilum, spotted fever group Rickettsia spp., typhus group Rickettsia spp., Coxiella burnetii, Bartonella spp., Orientia tsutsugamushi, Borrelia miyamotoi, Borrelia recurrentis and Leptospira spp. were systematically performed in all patients with undifferentiated NMF. We estimated the prevalence and predictive factors of DRI in returning travellers with undifferentiated NMF. RESULTS: Among 347 travellers with undifferentiated NMF, 106 (30·5%) were finally diagnosed with DRI. Only 57 (53·8%) of the 106 DRI infections were diagnosed by the standard of care. The main causes of DRI were: 55 (51·9%) Rickettsia spp., 16 (15·1%) C. burnetii; 15 (14·2%) Bartonella spp.; 13 (12·3%) Leptospira spp. and 10 (9·5%) A. phagocytophilum. The only predictive factor associated with DRI was presenting an eschar (aOR 39·52, 95%CI 4·85-322·18). Features of dengue such as retro-orbital pain (aOR 0·40, 95%CI 0·21-0·76) and neutropenia (aOR 0·41, 95%CI 0·21-0·79) were negatively associated with DRI. CONCLUSIONS: Although DRI are responsible for 30% of undifferentiated NMF cases in travellers, those are seldom recognized during the first clinical encounter. Empirical treatment with doxycycline should be considered in returning travellers with undifferentiated fever and negative tests for malaria and dengue, particularly when presenting severe illness, predictive factors for rickettsiosis or no features of dengue.

Epidemiological, Clinical, and Microbiological Characteristics in a Large Series of Patients Affected by Dermacentor-Borne-Necrosis-Erythema-Lymphadenopathy from a Unique Centre from Spain.

During recent decades, a tick-borne rickettsial syndrome, characterized by eschar and painful lymphadenopathy after Dermacentor marginatus-bite, has been described as an emerging rickettsiosis in Europe. Our group named it DEBONEL (Dermacentor-borne-necrosis-erythema-lymphadenopathy), regarding the vector and the main infection signs. Other groups called it TIBOLA (tick-borne-lymphadenophathy) and, later, SENLAT (scalp-eschar-and-neck-lymphadenopathy-after-tick-bite), expanding, in the latter, the etiological spectrum to other pathogens. Objective: To investigate the etiology of DEBONEL agents in our area, and to compare their epidemiological/clinical/microbiological characteristics. During 2001-2020, 216 patients clinically diagnosed of DEBONEL (the largest series from one center) in La Rioja (northern Spain) were examined. Rickettsia spp. were amplified in 14/104 (13.46%) blood samples, 69/142 (48.59%) eschar swabs, 7/7 (100%) biopsies, and 71/71 (100%) D. marginatus from patients. For samples in which Rickettsia was undetected, no other microorganisms were found. 'Candidatus Rickettsia rioja', Rickettsia slovaca, Rickettsia raoultii, and Rickettsia DmS1 genotype were detected in 91, 66, 4, and 3 patients, respectively. DEBONEL should be considered in patients with clinical manifestations herein described in areas associated to Dermacentor. The most frequently involved agent in our environment is 'Ca. R. rioja'. The finding of Rickettsia sp. DmS1 in ticks attached to DEBONEL patients suggests the implication of other rickettsia genotypes.

Excretion and viability of SARS-CoV-2 in feces and its association with the clinical outcome of COVID-19.

The main objective was to evaluate the viability of the SARS-CoV-2 viral particles excreted in stools. In addition, we aimed to identify clinical factors associated with the detection of SARS-CoV-2 RNA in feces, and to determine if its presence is associated with an unfavorable clinical outcome, defined as intensive care unit (ICU) admission and/or death. A prospective multicenter cohort study of COVID-19 adult patients, with confirmed SARS-CoV-2 infection by RT-PCR assay in nasopharyngeal (NP) swabs admitted to four hospitals in Spain, from March 2020 to February 2021. Sixty-two adult COVID-19 patients had stool samples collected at admission and/or during the follow up, with a total of 79 stool samples. SARS-CoV-2 RNA was detected in stool samples from 27 (43.5%) out of the 62 patients. Replicative virus, measured by the generation of cytopathic effect in cell culture and subsequent RT-PCR confirmation of a decrease in the Ct values, was not found in any of these stool samples. Fecal virus excretion was not associated with the presence of gastrointestinal symptoms, or with differences in the evolution of COVID-19 patients. Our results suggest that SARS-CoV-2 replicative capacity is null or very limited in stool samples, and thus, the fecal-oral transmission of SARS-CoV-2 as an alternative infection route is highly unlikely. In our study, the detection of SARS-CoV-2 RNA in feces at the beginning of the disease is not associated with any clinical factor nor with an unfavorable clinical outcome.

Serologic study of Bartonella sp. infection among human population of Southern Spain.

INTRODUCTION: The purpose of this study was to determine the prevalence of IgG antibodies against Bartonella sp. in a randomly selected sample from the population of the patients of North Sanitary District of Jaén. METHODS: We used a commercially available immunofluorescent test (Focus-Technology IFA Bartonella quintana and B. henselae test). RESULTS: Six hundred five healthy individuals were divided by sex into three age groups. We detected that 13.55% and 11.07% subjects were IgG seropositive to B. henselae and B. quintana, respectively. CONCLUSIONS: Our data show that the prevalence of both Bartonella species in Andalusia (Southern Spain) is relatively high. No statistical difference in the seropositivity was observed among these groups. In both cases, the IgG antibody titers ranged from 1/128 to 1/512.

Novel Genotypes of Nidicolous Argas Ticks and Their Associated Microorganisms From Spain.

The knowledge of the distribution, richness and epidemiological importance of soft ticks of the genus Argas is incomplete. In Spain, five Argas species have been recorded, including three ornitophilic nidicolous ticks, but their associated microorganisms remain unknown. This study aimed to investigate ticks from bird nests and their microorganisms. Ticks were collected extensively from natural cavities and nest-boxes used by European rollers (Coracias garrulus) and little owls (Athene noctua) in Southeastern and Central Spain. Ticks were morphologically and genetically identified and corresponding DNA/RNA tick extracts were analyzed [individually (n = 150) or pooled (n = 43)] using specific PCR assays for bacteria (Anaplasmataceae, Bartonella, Borrelia, Coxiella/Rickettsiella, and Rickettsia spp.), viruses (Flaviviruses, Orthonairoviruses, and Phenuiviruses), and protozoa (Babesia/Theileria spp.). Six Argas genotypes were identified, of which only those of Argas reflexus (n = 8) were identified to the species level. Two other genotypes were closely related to each other and to Argas vulgaris (n = 83) and Argas polonicus (n = 33), respectively. These two species have not been previously reported from Western Europe. Two additional genotypes (n = 4) clustered with Argas persicus, previously reported in Spain. The remaining genotype (n = 22) showed low sequence identity with any Argas species, being most similar to the African Argas africolumbae. The microbiological screening revealed infection with a rickettsial strain belonging to Rickettsia fournieri and Candidatus Rickettsia vini group in 74.7% of ticks, mainly comprising ticks genetically related to A. vulgaris and A. polonicus. Other tick endosymbionts belonging to Coxiella, Francisella and Rickettsiella species were detected in ten, one and one tick pools, respectively. In addition, one Babesia genotype, closely related to avian Babesia species, was found in one tick pool. Lastly, Anaplasmataceae, Bartonella, Borrelia, and viruses were not detected. In conclusion, five novel Argas genotypes and their associated microorganisms with unproven pathogenicity are reported for Spain. The re-use of nests between and within years by different bird species appears to be ideal for the transmission of tick-borne microorganisms in cavity-nesting birds of semiarid areas. Further work should be performed to clarify the taxonomy and the potential role of soft Argas ticks and their microorganisms in the epidemiology of zoonoses.

Incidence of human granulocytic anaplasmosis in returning travellers with fever.

BACKGROUND: Although tick-borne pathogens have been reported as an important cause of imported fever, the incidence of Anaplasma phagocytophilum, the causative agent of human granulocytic anaplasmosis (HGA), in travellers is unknown. METHODS: We conducted a prospective cohort study to investigate the aetiologies of fever in returning travellers (November 2017-July 2019). Polymerase chain reaction for msp2 gene amplification and indirect immunofluorescence assay for A. phagocitophilum were performed in all returning travellers with undifferentiated non-malarial fever. RESULTS: Among 141 travellers included, 8 patients were diagnosed with probable or confirmed HGA. The overall incidence rate of HGA was 19.9 cases/1000 person-week of travel. The main destination of travel was Asia, accounting for 62.5% patients with HGA. Co-infections were found in 37.5% of patients with HGA. CONCLUSIONS: Diagnosis of HGA and empirical treatment with doxycycline should be considered in travellers with fever.

What Does 16S rRNA Gene-Targeted Next Generation Sequencing Contribute to the Study of Infective Endocarditis in Heart-Valve Tissue?

Infective endocarditis (IE) is a severe and life-threatening disease. Identification of infectious etiology is essential for establishing the appropriate antimicrobial treatment and decreasing mortality. The aim of this study was to explore the potential utility of metataxonomics for improving microbiological diagnosis of IE. Here, next-generation sequencing (NGS) of the V3-V4 region of the 16S rRNA gene was performed in 27 heart valve tissues (18 natives, 5 intravascular devices, and 4 prosthetics) from 27 patients diagnosed with IE (4 of them with negative blood cultures). Metataxonomics matched with conventional diagnostic techniques in 24/27 cases (88.9%). The same bacterial family was assigned to 24 cases; the same genus, to 23 cases; and the same species, to 13 cases. In 22 of them, the etiological agent was represented by percentages > 99% of the reads and in two cases, by ~70%. Staphylococcus aureus was detected in a previously microbiological undiagnosed patient. Thus, microbiological diagnosis with 16S rRNA gene targeted-NGS was possible in one more sample than using traditional techniques. The remaining two patients showed no coincidence between traditional and 16S rRNA gene-targeted NGS microbiological diagnoses. In addition, 16S rRNA gene-targeted NGS allowed us to suggest coinfections that were supported by clinical data in one patient, and minority records also verified mixed infections in three cases. In our series, metataxonomics was valid for the identification of the causative agents, although more studies are needed before implementation of 16S rRNA gene-targeted NGS for the diagnosis of IE.

Detection of SARS-CoV-2 in pets living with COVID-19 owners diagnosed during the COVID-19 lockdown in Spain: A case of an asymptomatic cat with SARS-CoV-2 in Europe.

Pets from COVID-19 owners were screened for SARS-CoV-2 (April-May 2020). From 23 pets, an asymptomatic cat showed positive RT-qPCRs results from oropharyngeal swab (negative rectal swab). Remaining pets were negative. This suggests that cats can contract the virus from their infected owners and may act as potential hosts for SARS-CoV-2. Their role in carrying live or infectious viruses and disseminating them needs more investigation.

Serologic study of Bartonella sp. infection among human population of Southern Spain.

INTRODUCTION: The purpose of this study was to determine the prevalence of IgG antibodies against Bartonella sp. in a randomly selected sample from the population of the patients of North Sanitary District of Jaén. METHODS: We used a commercially available immunofluorescent test (Focus-Technology IFA Bartonella quintana and B. henselae test). RESULTS: Six hundred five healthy individuals were divided by sex into three age groups. We detected that 13.55% and 11.07% subjects were IgG seropositive to B. henselae and B. quintana, respectively. CONCLUSIONS: Our data show that the prevalence of both Bartonella species in Andalusia (Southern Spain) is relatively high. No statistical difference in the seropositivity was observed among these groups. In both cases, the IgG antibody titers ranged from 1/128 to 1/512.

Prevalence and molecular characterization of Rickettsia spp. in questing ticks from north-western Spain.

Tick-borne rickettsioses, most of them belonging to the spotted fever group (SFG), have been recognized as important emerging vector-borne zoonotic diseases. In order to determine the presence of Rickettsia spp. in questing ticks from north-western Spain, 1056 Ixodes ricinus, 19 Dermacentor marginatus, 17 Dermacentor reticulatus and one Ixodes acuminatus were processed. Rickettsia DNA was detected by PCR targeting rOmpA and rOmpB genes. A total of 219 (20.7%) I. ricinus, 19 (100%) D. marginatus and four D. reticulatus (23.5%) were positive. The prevalence was significantly higher in I. ricinus from coastal areas and in winter. Five species were identified: Rickettsia felis, Rickettsia monacensis, Rickettsia raoultii, Rickettsia slovaca and "Candidatus Rickettsia rioja". Our results reveal a significant presence of some pathogenic Rickettsia species in questing tick populations from this area which involves a noticeable risk of rickettsiosis. As R. raoultii, R. slovaca and "Ca. R. rioja" DNA were identified in I. ricinus, considered an unusual vector for these Rickettsia species, further studies are needed to unravel the role of that tick species in the maintenance and transmission of these three Rickettsia species in north-western Spain.

Exploring the bacteriome in anthropophilic ticks: To investigate the vectors for diagnosis.

OBJECTIVE: The aim of this study was to characterize the bacterial microbiome of hard ticks with affinity to bite humans in La Rioja (North of Spain). METHODS: A total of 88 adult ticks (22 Rhipicephalus sanguineus sensu lato, 27 Haemaphysalis punctata, 30 Dermacentor marginatus and 9 Ixodes ricinus) and 120 I. ricinus nymphs (CRETAV collection, La Rioja, Spain), representing the main anthropophilic species in our environment, were subjected to a metagenomic analysis of the V3-V4 region of the 16S rRNA gene using an Illumina MiSeq platform. Data obtained with Greengenes database were refined with BLAST. Four groups of samples were defined, according to the four tick species. RESULTS: Proteobacteria was the predominant phylum observed in all groups. Gammaproteobacteria was the most abundant class, followed by Alphaproteobacteria for R. sanguineus, H. punctata and D. marginatus but the relative abundance of reads for these classes was reversed for I. ricinus. This tick species showed more than 46% reads corresponding to 'not assigned' OTUs (Greengenes), and >97% of them corresponded to 'Candidatus Midichloriaceae' using BLAST. Within Rickettsiales, 'Candidatus Midichloria', Rickettsia, Ehrlichia, 'Candidatus Neoehrlichia' and Wolbachia were detected. I. ricinus was the most alpha-diverse species. Regarding beta-diversity, I. ricinus and H. punctata samples grouped according to their tick species but microbial communities of some R. sanguineus and D. marginatus specimens clustered together. CONCLUSIONS: The metagenomics approach seems useful to discover the spectrum of tick-related bacteria. More studies are needed to identify and differentiate bacterial species, and to improve the knowledge of tick-borne diseases in Spain.

Borrelia miyamotoi: Should this pathogen be considered for the diagnosis of tick-borne infectious diseases in Spain?

INTRODUCTION: Borrelia miyamotoi is a tick-borne pathogen belonging to the relapsing fever group. It had not been reported from Spain, but its wide distribution and the presence of the tick-vector (Ixodes ricinus) made us suspect its circulation. The aim of this study was to investigate the presence of Borrelia spp. in I. ricinus in Spain. METHODS: A total of 652 I. ricinus nymphs collected in northern Spain were processed. The DNA was extracted using incubations with ammonium hydroxide. Borrelia spp. DNA was amplified using Borrelia-specific PCR assays (glpQ, 16S rRNA and flagellin genes). RESULTS: B. miyamotoi was amplified in 4 specimens, and Borrelia burgdorferi sensu lato in 27 (8 Borrelia afzelii, 7 Borrelia garinii, 8 Borrelia lusitaniae, 3 Borrelia valaisiana and 1 B. burgdorferi sensu stricto). CONCLUSION: B. miyamotoi should be considered in the differential diagnoses of patients with confirmed or suspected tick-bite in Spanish endemic areas for Lyme disease.

Epidemiology of Spotted Fever Group Rickettsioses and Acute Undifferentiated Febrile Illness in Villeta, Colombia.

Etiology of acute undifferentiated febrile syndrome (AUFS) is often unknown, leading to inaccurate diagnosis and treatment. Villeta town has been identified as an endemic area for spotted fever group (SFG) rickettsioses but little is known about possible amplifier hosts and other Rickettsia species different from Rickettsia rickettsii. Besides, few studies have approached other AUFS etiologies in the region. We investigated the role of dengue, leptospirosis, rickettsioses, human anaplasmosis, and Q fever as possible causes of AUFS in patients from Villeta. Sera specimens and ticks from animals as well as ticks from vegetation were studied for the presence of different Rickettsia spp. Among 104 sera from patients with AUFS, 16.4%, 24.0%, and 2.9% patients seroconverted to dengue, Leptospira, and SFG Rickettsia, respectively, with a case of probable coinfection or cross-reaction with Anaplasma phagocytophilum. None of the samples were reactive for Coxiella burnetii. Sera samples from 74 horses, 118 dogs, and 62 bovines were collected and showed 33.8%, 14.4%, and 50.0% of seroprevalence for SFG Rickettsia, respectively. A total of 1,287 ixodid ticks were collected from animals/vegetation and processed in pools for polymerase chain reaction. Among them, 1.7% was positive for Rickettsia genes, and Rickettsia amblyommii, R. rickettsii, and Rickettsia spp. were found. These results confirm the circulation of dengue, different SFG Rickettsia species and the relevance of other etiologies like leptospirosis and human anaplasmosis. Further studies must identify different epidemiological variables to establish proper surveillance and control programs.

Molecular (ticks) and serological (humans) study of Crimean-Congo hemorrhagic fever virus in the Iberian Peninsula, 2013-2015.

INTRODUCTION: Crimean-Congo hemorrhagic fever (CCHF) is a viral disease, mainly transmitted through tick bite, of great importance in Public Health. In Spain, Crimean-Congo hemorrhagic fever virus (CCHFV) was detected for the first time in 2010 in Hyalomma lusitanicum ticks collected from deer in Cáceres. The aim of this study was to investigate the presence of CCHFV in ticks from Cáceres, and from other Spanish areas, and to evaluate the presence of antibodies against the virus in individuals exposed to tick bites. METHODS: A total of 2053 ticks (1333 Hyalomma marginatum, 680 H. lusitanicum and 40 Rhipicephalus bursa) were analyzed using molecular biology techniques (PCR) for CCHFV detection. The determination of specific IgG antibodies against CCHFV in 228 serum samples from humans with regular contact with ticks (at risk of acquiring the infection) was performed by indirect immunofluorescence assay. RESULTS: The CCHFV was not amplified in ticks, nor were antibodies against the virus found in the serum samples analyzed. CONCLUSION: The absence of the CCHFV in the ticks studied and the lack of antibodies against the virus in individuals exposed to tick bites would seem to suggest a low risk of acquisition of human infection by CCHFV in Spain.

Guidelines for the Detection of Rickettsia spp.

The genus Rickettsia (Rickettsiales: Rickettsiaceae) includes Gram-negative, small, obligate intracellular, nonmotile, pleomorphic coccobacilli bacteria transmitted by arthropods. Some of them cause human and probably also animal disease (life threatening in some patients). In these guidelines, we give clinical practice advices (microscopy, serology, molecular tools, and culture) for the microbiological study of these microorganisms in clinical samples. Since in our environment rickettsioses are mainly transmitted by ticks, practical information for the identification of these arthropods and for the study of Rickettsia infections in ticks has also been added.

Isolation and maintenance of Rickettsia raoultii in a Rhipicephalus sanguineus tick cell line.

Rickettsia raoultii, a member of the spotted fever group rickettsiae, has been implicated in cases of DEBONEL/TIBOLA/SENLAT, and has been detected in Dermacentor spp. and Rhipicephalus pumilio ticks by PCR. R. raoultii has been isolated in mammalian and tick cell lines. This study aimed to isolate R. raoultii from Spanish Dermacentor marginatus in tick cell lines. A single adult D. marginatus collected from vegetation in La Rioja (Northen Spain) in October 2012 was surface-sterilised, triturated and aliquots of the homogenate were inoculated into a panel of tick cell lines derived from embryonic Rhipicephalus sanguineus, Rhipicephalus evertsi and Ixodes ricinus. Cultures were maintained at 28 °C with weekly medium changes and checked by Gimenez stain for Rickettsia-like intracellular organisms. After 50 days of incubation, intracellular Rickettsia-like organisms were observed in the R. sanguineus cell line RML-RSE using Gimenez stain. PCR assays and sequencing of fragments of 16S RNA, ompB and ompA genes in DNA extracted from the culture suspension showed 100% identity with R. raoultii. Growth of intracellular microorganisms was not observed in preparations of the other tick cell lines. In conclusion, the tick cell line RML-RSE is a useful system for the isolation and maintenance of R. raoultii.

Nuclear Magnetic Resonance (NMR) as a tool for the study of the metabolism of Rickettsia slovaca.

Rickettsial infections are caused by intracellular bacteria. They do not grow in standard culture media so there are limitations in routine practice to study their metabolism. Nuclear Magnetic Resonance (NMR) spectroscopy is used for identification of metabolites in biological samples. Vero cells infected with Rickettsia slovaca as well as uninfected cells were monitored by (1)H NMR showing the presence of ethanol and lactic acid. As no differences were observed, labeled compounds were added into cultures. When D-[1-13C]glucose was monitored by (13)C NMR no differences among infected and uninfected cells were observed in metabolic profiles. Glucose was transformed into ethanol in all cultures. Monitored experiments carried out with [2-13C]glycine showed differences between infected and uninfected cell cultures spectra. Glycine was partially transformed into serine, but the amount of the serine formed was larger in those infected. Moreover, L-[2-13C]leucine, L-[1-13C]isoleucine and L-[15N]tyrosine were evaluated. No differences among infected and uninfected cells were observed in the metabolic profiles when tyrosine and leucine were monitored. The amino acid L-[1-13C]isoleucine exhibited different metabolism in presence of the R. slovaca, showing a promising behavior as biomarker. In this work we focused on finding one or more compounds that could be metabolized specifically by R. slovaca and could be used as an indicator of its activity.