The evolution of intramitochondriality in Midichloria bacteria.

Midichloria spp. are intracellular bacterial symbionts of ticks. Representatives of this genus colonise mitochondria in the cells of their hosts. To shed light on this unique interaction we evaluated the presence of an intramitochondrial localization for three Midichloria in the respective tick host species and generated eight high-quality draft genomes and one closed genome, showing that this trait is non-monophyletic, either due to losses or multiple acquisitions. Comparative genomics supports the first hypothesis, as the genomes of non-mitochondrial symbionts are reduced subsets of those capable of colonising the organelles. We detect genomic signatures of mitochondrial tropism, including the differential presence of type IV secretion system and flagellum, which could allow the secretion of unique effectors and/or direct interaction with mitochondria. Other genes, including adhesion molecules, proteins involved in actin polymerisation, cell wall and outer membrane proteins, are only present in mitochondrial symbionts. The bacteria could use these to manipulate host structures, including mitochondrial membranes, to fuse with the organelles or manipulate the mitochondrial network.

Trombiculiasis in a Dog with Severe Neurologic Disorders, Spain.

Chiggers, the larvae of trombiculid mites, parasitize a wide variety of terrestrial vertebrates worldwide. Their bites cause seasonal trombiculiasis in humans and animals. Affected canines can have a variety of digestive and systemic clinical signs. We describe a case of canine trombiculiasis in a dog exhibiting severe neurologic symptoms.

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.

Novel Candidatus rickettsia species detected in nostril tick from human, Gabon, 2014.

We report the identification of a nymphal nostril tick (Amblyomma sp.) from a national park visitor in Gabon and subsequent molecular detection and characterization of tickborne bacteria. Our findings provide evidence of a potentially new Rickettsia sp. circulating in Africa and indicate that tick bites may pose a risk to persons visiting parks in the region.

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.

Crimean-Congo hemorrhagic fever virus in ticks from migratory birds, Morocco.

Crimean-Congo hemorrhagic fever virus was detected in ticks removed from migratory birds in Morocco. This finding demonstrates the circulation of this virus in northwestern Africa and supports the hypothesis that the virus can be introduced into Europe by infected ticks transported from Africa by migratory birds.

Usefulness of rickettsial PCR assays for the molecular diagnosis of human rickettsioses.

BACKGROUND: The effectiveness of PCR methods to amplify rickettsiae from clinical samples has still not been evaluated. Our aim was to determine the sensitivity and usefulness for Rickettsia species identification by PCR methods, targeting 16S rDNA, htrA, gltA, ompA, and ompB genes for molecular diagnosis of rickettsioses. METHODS: A total of 72 clinical samples (EDTA-blood, skin biopsies and ticks) taken from 52 patients in the early phase of the illness with PCR-confirmed rickettsioses were included. Single [16S rDNA, gltA (5' end), and htrA genes] and sequential (nested or semi-nested) PCR assays [ompB, gltA (central region) and ompA genes] were performed. RESULTS: For single-stage PCR assays, the greatest sensitivity (33.3%) was obtained using the gltA (5' end), while for sequential assays, the most sensitive results were obtained using the ompB assay (83.3%). The highest sensitivity (100%) was achieved using the three sequential PCRs. The ompA PCR method was the most reliable for identifying Rickettsia species, according to clinical features. CONCLUSIONS: PCR-based amplification methods are useful rickettsial diagnostic tools in the early phase of the illness. The three sequential PCR assays here investigated (ompB, gltA and ompA) appear to be useful tools for molecular diagnosis of rickettsioses. ompB PCR assay is effective for primary screening, since it detects a high percentage of positive samples. ompA assay is the most useful method to identify a Rickettsia species in human pathology. Nevertheless, epidemiology, clinical symptoms and the vector involved in the infection have to be taken into account for the diagnosis of rickettsioses.

MALDI-TOF MS as a tick identification tool in a tertiary hospital in Spain.

In Spain, as in other countries, the spectrum of tick-borne diseases and their number have increased in recent years. The tick identification, at species level, can be challenging outside research centers although this information is very usufull for decisions making. The performance of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) in tick identification of specimens collected from patients have been seldomly reported. The aim of the present study was to desing a protein-extraction protocol and build a tick-legs reference spectra. This protocol was then validated using specimens from both patients and non-patient sources. Nine species of ticks that usually bites humans in Spain were included: Dermacentor marginatus, Dermacentor reticulatus, Haemaphysalis punctata, Hyalomma lusitanicum, Hyalomma marginatum, Ixodes ricinus, Rhipicephalus bursa, Rhipicephalus pusillus and Rhipicephalus sanguineus sensu lato. Other less-frequent biting species were also included: Haemaphysalis inermis, Haemaphysalis concinna, Hyalomma scupense, Ixodes frontalis, Ixodes hexagonus, and Argas sp. specimens were identified by PCR and sequencing of a fragment of the 16S rRNA gene of ticks. In the tests performed with non-patient collected specimens, a 100% correlation was observed between molecular methods and MS, while in the tests performed with ticks collected from patients a 92.59% correlation was observed. Misidentification was observed only in two of I. ricinus nymphs (identified as Ctenocephalides felis). Therefore, mass- spectrometry can be confidently used as a tick identification tool in a hospital setting for the rapid identification of tick vectors.

Role of birds in dispersal of etiologic agents of tick-borne zoonoses, Spain, 2009.

We amplified gene sequences from Anaplasma phagocytophilum, Borrelia garinii, B. valaisiana, B. turdi, Rickettsia monacensis, R. helvetica, R. sibirica sibirica, and Rickettsia spp. (including Candidatus Rickettsia vini) in ticks removed from birds in Spain. The findings support the role of passerine birds as possible dispersers of these tick-borne pathogens.

Old zoonotic agents and novel variants of tick-borne microorganisms from Benguela (Angola), July 2017.

BACKGROUND: Ticks and tick-borne diseases constitute a real threat for the livestock industry, which is increasing in Angola. In addition, ticks are vectors of zoonoses of public health concern, and scarce information is available from this country. In an effort to contribute to the prevention of zoonotic infectious diseases affecting humans and animals, the molecular screening of certain tick-related microorganisms collected on cattle in Angola was performed under a 'One Health' scope. METHODS: Ticks collected from cattle in Cubal (Benguela Province, Angola) in July 2017 were analysed in pools using specific PCR assays for bacteria (Rickettsia, Anaplasmataceae, Borrelia, Coxiella and Spiroplasma) and protozoa (Theileria and Babesia) detection. RESULTS: A total of 124 tick specimens were grouped in 25 pools (two Amblyomma variegatum, three Hyalomma truncatum, 16 Rhipicephalus decoloratus, two Rhipicephalus duttoni, one Rhipicephalus evertsi mimeticus and one Rhipicephalus sp.). The amplified microorganisms were (pools): Rickettsia africae (two A. variegatum and one R. decoloratus), Rickettsia aeschlimannii (three H. truncatum), Ehrlichia spp. (six R. decoloratus), Coxiella spp. (all but H. truncatum), Francisella sp. (one H. truncatum), Spiroplasma sp. closely related to Spiroplasma ixodetis (three R. decoloratus), Babesia bigemina (two R. decoloratus) and Babesia spp. (two A. variegatum). The obtained nucleotide sequences from Ehrlichia spp., two Coxiella genotypes (from R. duttoni and Rhipicephalus sp.), Francisella sp. and Babesia spp. (from A. variegatum) reached low identities with known genetically characterized species. CONCLUSIONS: This study demonstrates the circulation in Angola of the pathogen R. aeschlimannii and potential novel tick-related microorganisms belonging to Ehrlichia, Coxiella, Francisella, Spiroplasma and Babesia spp. and corroborates the presence of R. africae and B. bigemina. Our results should be considered in developing protocols for the management of fever of unknown origin and for veterinary practices. Further studies are required to evaluate the risk of tick-borne diseases in Angola.

New Cell Lines Derived from European Tick Species.

Tick cell lines are important tools for research on ticks and the pathogens they transmit. Here, we report the establishment of ten new cell lines from European ticks of the genera Argas, Dermacentor, Hyalomma, Ixodes and Rhipicephalus originating from Germany and Spain. For each cell line, the method used to generate the primary culture, a morphological description of the cells and species confirmation by sequencing of the partial 16S rRNA gene are presented. Further molecular analysis of the two new Ixodes ricinus cell lines and three existing cell lines of the same species revealed genetic variation between cell lines derived from ticks collected in the same or nearby locations. Collectively, these new cell lines will support research into a wide range of viral, bacterial and protozoal tick-borne diseases prevalent in Europe.

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.

Epidemiological Aspects of Crimean-Congo Hemorrhagic Fever in Western Europe: What about the Future?

Crimean-Congo hemorrhagic fever virus (CCHFV) is an arthropod-borne virus (arbovirus), mainly transmitted by ticks, belonging to the genus Orthonairovirus (family Nairoviridae, order Bunyavirales). CCHFV causes a potentially severe, or even fatal, human disease, and it is widely distributed in Africa, Asia, eastern Europe and, more recently, in South-western Europe. Until a few years ago, no cases of Crimean-Congo hemorrhagic fever (CCHF) had been reported in western Europe, with the exception of several travel-associated cases. In 2010, the CCHFV was reported for the first time in South-western Europe when viral RNA was obtained from Hyalomma lusitanicum ticks collected from deer in Cáceres (Spain). Migratory birds from Africa harboring CCHFV-infected ticks and flying to Spain appear to have contributed to the establishment of the virus (genotype III, Africa-3) in this country. In addition, the recent findings in a patient and in ticks from deer and wild boar of viral sequences similar to those from eastern Europe (genotype V, Europe-1), raise the possibility of the introduction of CCHFV into Spain through the animal trade, although the arrival by bird routes cannot be ruled out (Africa-4 has been also recently detected). The seropositive rates of animals detected in regions of South-western Spain suggest an established cycle of tick-host-tick in certain areas, and the segment reassortment detected in the sequenced virus from one patient evidences a high ability to adaptation of the virus. Different ixodid tick genera can be vectors and reservoirs of the virus, although Hyalomma spp. are particularly relevant for its maintenance. This tick genus is common in Mediterranean region but it is currently spreading to new areas, partly due to the climate change and movement of livestock or wild animals. Although to a lesser extent, travels with our pets (and their ticks) may be also a factor to be considered. As a consequence, the virus is expanding from the Balkan region to Central Europe and, more recently, to Western Europe where different genotypes are circulating. Thus, seven human cases confirmed by molecular methods have been reported in Spain from 2016 to August 2020, three of them with a fatal outcome. A One Health approach is essential for the surveillance of fauna and vector populations to assess the risk for humans and animals. We discuss the risk of CCHFV causing epidemic outbreaks in Western Europe.

Spiroplasma Isolated From Third-Generation Laboratory Colony Ixodes persulcatus Ticks.

Spiroplasma are vertically-transmitted endosymbionts of ticks and other arthropods. Field-collected Ixodes persulcatus have been reported to harbour Spiroplasma, but nothing is known about their persistence during laboratory colonisation of this tick species. We successfully isolated Spiroplasma from internal organs of 6/10 unfed adult ticks, belonging to the third generation of an I. persulcatus laboratory colony, into tick cell culture. We screened a further 51 adult male and female ticks from the same colony for presence of Spiroplasma by genus-specific PCR amplification of fragments of the 16S rRNA and rpoB genes; 100% of these ticks were infected and the 16S rRNA sequence showed 99.8% similarity to that of a previously-published Spiroplasma isolated from field-collected I. persulcatus. Our study shows that Spiroplasma endosymbionts persist at high prevalence in colonised I. persulcatus through at least three generations, and confirms the usefulness of tick cell lines for isolation and cultivation of this bacterium.

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.

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.

Bacterial Pathogens and Symbionts Harboured by Ixodes ricinus Ticks Parasitising Red Squirrels in the United Kingdom.

Red squirrels (Sciurus vulgaris) are native to most of Eurasia; in much of the United Kingdom, they have been supplanted by the non-native grey squirrel, and are considered an endangered species. Very little is known about the range of tick-borne pathogens to which UK red squirrels are exposed. As part of trap-and-release surveys examining prevalence of Mycobacterium spp. in red squirrel populations on two UK islands, Ixodes ricinus ticks were removed from squirrels and PCR screened for Borrelia spp., intracellular arthropod-borne bacteria and the parasitic wasp Ixodiphagus hookeri. At both sites, the most commonly encountered tick-transmitted bacterium was Borrelia burgdorferi sensu lato (overall minimum prevalence 12.7%), followed by Anaplasma phagocytophilum (overall minimum prevalence 1.6%). Single ticks infected with Spiroplasma were found at both sites, and single ticks infected with Borrelia miyamotoi or an Ehrlichia sp. at one site. Ticks harbouring Wolbachia (overall minimum prevalence 15.2%) were all positive for I. hookeri. Our study shows that UK red squirrels are potentially exposed to a variety of bacterial pathogens via feeding ticks. The effects on the health and survival of this already vulnerable wildlife species are unknown, and further studies are needed to evaluate the threat posed to red squirrels by Borrelia and other tick-borne pathogens.

A dual endosymbiosis supports nutritional adaptation to hematophagy in the invasive tick Hyalomma marginatum.

Many animals are dependent on microbial partners that provide essential nutrients lacking from their diet. Ticks, whose diet consists exclusively on vertebrate blood, rely on maternally inherited bacterial symbionts to supply B vitamins. While previously studied tick species consistently harbor a single lineage of those nutritional symbionts, we evidence here that the invasive tick Hyalomma marginatum harbors a unique dual-partner nutritional system between an ancestral symbiont, Francisella, and a more recently acquired symbiont, Midichloria. Using metagenomics, we show that Francisella exhibits extensive genome erosion that endangers the nutritional symbiotic interactions. Its genome includes folate and riboflavin biosynthesis pathways but deprived functional biotin biosynthesis on account of massive pseudogenization. Co-symbiosis compensates this deficiency since the Midichloria genome encompasses an intact biotin operon, which was primarily acquired via lateral gene transfer from unrelated intracellular bacteria commonly infecting arthropods. Thus, in H. marginatum, a mosaic of co-evolved symbionts incorporating gene combinations of distant phylogenetic origins emerged to prevent the collapse of an ancestral nutritional symbiosis. Such dual endosymbiosis was never reported in other blood feeders but was recently documented in agricultural pests feeding on plant sap, suggesting that it may be a key mechanism for advanced adaptation of arthropods to specialized diets.

Isolation and partial characterisation of a novel Trypanosoma from the tick Ixodes ricinus.

Trypanosomes have long been recognised as being amongst the most important protozoan parasites of vertebrates, from both medical and veterinary perspectives. Whilst numerous insect species have been identified as vectors, the role of ticks is less well understood. Here we report the isolation and partial molecular characterisation of a novel trypanosome from questing Ixodes ricinus ticks collected in Slovakia. The trypanosome was isolated in tick cell culture and then partially characterised by microscopy and amplification of fragments of the 18S rRNA and 24Sα rDNA genes. Analysis of the resultant sequences suggests that the trypanosome designated as Trypanosoma sp. Bratislava1 may be a new species closely related to several species or strains of trypanosomes isolated from, or detected in, ticks in South America and Asia, and to Trypanosoma caninum isolated from dogs in Brazil. This study highlights the potential involvement of ixodid ticks in the epidemiology of trypanosomes, as well as the use of tick cell lines for isolation of such tick-borne protozoa. Further studies are required to investigate the epidemiology, transmission and life cycle of this putative novel species.

Isolation of Candidatus Rickettsia vini from Belgian Ixodes arboricola ticks and propagation in tick cell lines.

Candidatus Rickettsia vini was originally detected in Ixodes arboricola ticks from Spain, and subsequently reported from several other Western Palearctic countries including Belgium. Recently, the bacterium was isolated in mammalian (Vero) cell culture from macerated male I. arboricola from Czech Republic, but there have been no reports of propagation in tick cells. Here we report isolation in a tick cell line of three strains of Ca. R. vini from I. arboricola collected from nests of great tits (Parus major) in Belgium. Internal organs of one male and two engorged female ticks were dissected aseptically, added to cultures of the Rhipicephalus microplus cell line BME/CTVM23 and incubated at 28 °C. Rickettsia-like bacteria were first seen in Giemsa-stained cytocentrifuge smears between 2 and 15 weeks later. Two of the isolates grew rapidly, destroying the tick cells within 2-4 weeks of onward passage in BME/CTVM23 cells, while the third isolate grew much more slowly, only requiring subculture at 4-5-month intervals. PCR amplification of bacterial 16S rRNA and Rickettsia gltA, sca4, ompB, ompA and 17-kDa genes revealed that all three isolates were Ca. R. vini, with 100 % identity to each other and to published Ca. R. vini sequences from other geographical locations. Transmission electron microscopy revealed typical single Rickettsia bacteria in the cytoplasm of BME/CTVM23 cells. The Ca. R. vini strain isolated from the male I. arboricola tick, designated Boshoek1, was tested for ability to grow in a panel of Ixodes ricinus, Ixodes scapularis and R. microplus cell lines and in Vero cells. The Boshoek1 strain grew rapidly, causing severe cytopathic effect, in the R. microplus line BME26, the I. ricinus line IRE11 and Vero cells, more slowly in the I. ricinus line IRE/CTVM19, possibly established a low-level infection in the I. ricinus line IRE/CTVM20, and failed to infect cells of any of four I. scapularis lines over a 12-week observation period. This study confirmed the applicability of the simple tick organ-cell line co-cultivation technique for isolation of tick-borne Rickettsia spp. using BME/CTVM23 cells.