Cristina Cervera-Acedo

Background: Leishmaniosis is a zoonotic disease caused by protozoan parasites of the Leishmania genus, primarily transmitted through the bites of infected female phlebotomine sand flies. Leishmania infantum is the most prevalent Leishmania species in the Mediterranean Region, affecting both humans and animals, mainly dogs. Integrated surveillance strategies, including xenomonitoring, are essential for early detection and cost-effective control in endemic areas. In 2021, an increase in human leishmaniosis cases was reported in the Region of Murcia (Spain), prompting an intensified entomological surveillance effort aimed at assessing Leishmania prevalence in vector populations. Phlebotomine sand flies were collected and analysed to determine the circulation of Leishmania spp. in the area. Subsequently, these field-collected samples were used to evaluate the diagnostic performance of a commercial real-time PCR (qPCR) kit designed for the detection of Leishmania DNA. Methods: A comparative–retrospective analysis was carried out at the Parasitology Laboratory of the Department of Animal Pathology, University of Zaragoza. A total of 314 DNA extracts from pooled female sand fly samples, collected in urban and peri-urban areas of 29 municipalities in the Region of Murcia near recently reported human leishmaniosis cases (2021–2022), were analysed. Sampling was conducted in June and September 2023 using CO ₂ -baited light traps to investigate the presence of Leishmania spp. in sand flies. The performance of the VIASURE qPCR assay (CerTest Biotec, Zaragoza, Spain) was assessed against a structured reference method consisting of an in-house qPCR assay, conventional PCR followed by Sanger sequencing and a SYBR Green qPCR used to resolve discordant results. Results: Of the 314 pooled samples analysed, 21 tested positive for Leishmania spp. using the reference methods, while the VIASURE assay identified 23 Leishmania spp.-positive pooled samples. After resolving discrepancies, the obtained analytical sensitivity and specificity were 0.95 (95% confidence interval [CI] 0.76–0.99) and 0.99 (95% CI 0.97–0.99), respectively. Conclusions: The commercial VIASURE qPCR assay showed high concordance with established molecular methods and demonstrated reliable performance for detecting Leishmania DNA in sand fly vectors. To our knowledge, this is the first study to specifically validate a commercially available qPCR kit in phlebotomine sand flies. These findings support its potential utility in entomological surveillance programs and public health interventions in leishmaniosis-endemic regions.

Candidatus Rickettsia colombiensis is a new candidate species of Rickettsiae spotted fever group that have been isolated only from ticks. The pathogenicity of Ca. R. colombiensis to human and animals is unknown. This study evaluated the pathogenic potential of Ca. R. colombiensis in Syrian hamsters and assessed the cross-reactivity between Ca. R. colombiensis and other Rickettsia in human and hamster sera. Shell vial technique was employed to isolate Ca. R. colombiensis. Subsequently, five male Syrian hamsters were inoculated intraperitoneally (IP) and five intradermally (ID) with 1 × 10⁶ Vero cells infected with Ca. R. colombiensis. One control hamster was used in each group. The health status was assessed daily, and necropsies were performed. Serum samples were tested by indirect immunofluorescence and tissues were processed by qPCR and histological stains. All Syrian hamsters remained healthy during the trial. No histopathological damages associated with rickettsial infection were observed. No Rickettsial DNA was detected in tissues. Syrian hamsters showed IgG antibody titers ranging from 1:64 to 1:1024. Control hamsters were negative. Regarding human sera, 56% (84/150) had IgG cross-reactivity antibodies against Ca. R. colombiensis. Subsequently, in a selected subset of 30 sera with moderate to high titers, all samples reacted with Ca. R. colombiensis antigen. Under specific conditions of this study, Ca. R. colombiensis did not behave as a highly virulent pathogen in the hamster model, although all infected Syrian hamsters developed IgG antibodies responses. Regarding cross-reactivity, it is possible to serologically diagnose rickettsial infection using Ca. R. colombiensis as an antigen.

Wildlife is an important source of emerging zoonotic agents, including tick‐borne ones. Wild carnivores such as mink are commonly parasitized by ticks, which are vectors and reservoirs of zoonotic diseases. Besides the importance of these arthropods as potential sources of diseases in mink, and the role of these mammals as reservoirs of infectious diseases, scarce studies of microorganisms of ticks from mink have been performed. In the present work, ticks collected from mink (European mink [ Mustela lutreola ] and American mink [ Neogale vison ]) from 2007 to 2021 in the North of Spain, and their associated microorganisms (bacteria, protozoan, and viruses), were studied. A total of 916 specimens (154 Ixodes acuminatus , 761 Ixodes hexagonus , and one Rhipicephalus sanguineus sensu lato) were processed in 165 pools (31 I. acuminatus , 133 I. hexagonus , and one R. sanguineus s.l.). The microorganisms detected in I. acuminatus pools were Ehrlichia sp. (8), Neoehrlichia mikurensis (4), Coxiella spp. (23), Rickettsiella spp. (7), and Ixovirus spp. (3). In I. hexagonus pools, Coxiella spp. (131), Rickettsiella spp. (5), Hepatozoon martis (5), and Ixovirus spp. (6) were amplified. Infection with Coxiella spp. was found in the R. sanguineus s.l. specimen. Mink are involved in the epidemiology of tick‐borne microorganisms, including important pathogens. The role of these tick species as vectors and mink as reservoirs of these microorganisms should be further investigated.

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.

Leishmaniasis, endemic in Spain, displays rising incidence rates, notably in the Region of Murcia. Sandfly surveillance and Leishmania screening in 2021 uncovered Leishmania infantum and L. adleri . While current Leishmania diagnostic methods primarily target L. infantum , our study underscores the importance of broader Leishmania species surveillance for accurate diagnosis and understanding of disease dynamics. The detection of L. adleri , previously undescribed in Europe, highlights the need for expanded research to investigate its presence and implications for human and animal health.

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.

During April-May 2020, the presence of respiratory syndrome coronavirus 2 (SARS-CoV-2) in pets living with coronavirus disease 2019 (COVID-19) owners was analyzed. From 23 pets, a cat without clinical symptoms showed positive results for SARS-CoV-2 in oropharyngeal swab using three RT-qPCR assays (negative rectal swab). SARS-CoV-2 was not detected in the remaining pets. Our finding suggests that cats may act as asymptomatic dispersers of SARS-CoV-2, although viral transmission from animals to humans seems unlikely.

Alport syndrome is an inherited renal disorder characterized by glomerular basement membrane lesions with hematuria, proteinuria and frequent hearing defects and ocular abnormalities. The disease is associated with mutations in genes encoding α3, α4, or α5 chains of type IV collagen, namely COL4A3 and COL4A4 in chromosome 2 and COL4A5 in chromosome X. In contrast to the well-known X-linked and autosomal recessive phenotypes, there is very little information about the autosomal dominant. In view of the wide spectrum of phenotypes, an exact diagnosis is sometimes difficult to achieve. We investigated a Spanish family with variable phenotype of autosomal dominant Alport syndrome using clinical, histological, and genetic analysis. Mutational analysis of COL4A3 and COL4A4 genes showed a novel heterozygous mutation (c. 998G > A; p.G333E) in exon 18 of the COL4A3 gene. Among relatives carrying the novel mutation, the clinical phenotype was variable. Two additional COL4A3 mutations were found, a Pro-Leu substitution in exon 48 (p.P1461L) and a Ser-Cys substitution in exon 49 (p.S1492C), non-pathogenics alone. Carriers of p.G333E and p.P1461L or p.S1492C mutations in COL4A3 gene appear to be more severely affected than carriers of only p.G333E mutation, and the clinical findings has an earlier onset. In this way, we could speculate on a synergistic effect of compound heterozygosity that could explain the different phenotype observed in this family.

Rubinstein-Taybi syndrome (RSTS; OMIM #180849, #613684) is a rare autosomal dominant genetic condition characterized by broad thumbs and halluces, facial dysmorphism, short stature and variable degree of intellectual disability. RSTS is associated with mutations in CREBBP and EP300 genes in 50–60% and 5–8% of cases, respectively. The majority of cases are de novo heterozygous mutations. Here we describe a familial RSTS case, associated with a novel EP300 mutation. The proband was a 9 years old female, with mild learning difficulties. Her mother, who also had learning difficulties, was found to have short and broad thumbs. MLPA and panel-based NGS of CREBBP and EP300 were performed. A novel heterozygous frameshift mutation in exon 31 of the EP300 gene (c.7222_7223del; p.(Gln2408Glufs*39)) was found in both. This case represents the first case of inherited EP300-RSTS. The location of the frameshift deletion not affecting HAT domain and PHD finger, could explain the mild phenotype and the well-preserved intelligence. These patients are mildly affected, and this case highlights the possible missed diagnosis. We would recommend molecular testing of apparently healthy parents, and in the case of inherited mutations, of all adult first degree relatives at risk.