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The taxonomy of the order Piroplasmida, which includes a number of clinically and economically relevant organisms, is a hotly debated topic amongst parasitologists. Three genera (Babesia, Theileria, and Cytauxzoon) are recognized based on parasite life cycle characteristics, but molecular phylogenetic analyses of 18S sequences have suggested the presence of five or more distinct Piroplasmida lineages. Despite these important advancements, a few studies have been unable to define the taxonomic relationships of some organisms (e.g. C. felis and T. equi) with respect to other Piroplasmida. Additional evidence from mitochondrial genome sequences and synteny should aid in the inference of Piroplasmida phylogeny and resolution of taxonomic uncertainties. In this study, we have amplified, sequenced, and annotated seven previously uncharacterized mitochondrial genomes (Babesia canis, Babesia vogeli, Babesia rossi, Babesia sp. Coco, Babesia conradae, Babesia microti-like sp., and Cytauxzoon felis) and identified additional ribosomal fragments in ten previously characterized mitochondrial genomes. Phylogenetic analysis of concatenated mitochondrial and 18S sequences as well as cox1 amino acid sequence identified five distinct Piroplasmida groups, each of which possesses a unique mitochondrial genome structure. Specifically, our results confirm the existence of four previously identified clades (B. microti group, Babesia sensu stricto, Theileria equi, and a Babesia sensu latu group that includes B. conradae) while supporting the integration of Theileria and Cytauxzoon species into a single fifth taxon. Although known biological characteristics of Piroplasmida corroborate the proposed phylogeny, more investigation into parasite life cycles is warranted to further understand the evolution of the Piroplasmida. Our results provide an evolutionary framework for comparative biology of these important animal and human pathogens and help focus renewed efforts toward understanding the phylogenetic relationships within the group.

Background Cytauxzoon spp. infection is believed to be a newly emerging tick-borne disease in felids in Europe, with three species of the haemoparasite having recently been differentiated in wild felids. In Switzerland, rare infections have been documented in domestic cats in the west and northwest of the country, the first of which was in 2014. The aims of the present study were: (i) to characterize a Cytauxzoon spp. hotspot in domestic cats in central Switzerland; (ii) to elucidate the geographic distribution of Cytauxzoon spp. in domestic cats in Switzerland; (iii) to assess suspected high-risk populations, such as stray and anaemic cats; and (iv) to investigate the newly emerging nature of the infection. Cytauxzoon spp. were further differentiated using mitochondrial gene sequencing. Methods The overall study included samples from 13 cats from two households in central Switzerland (study A), 881 cats from all regions of Switzerland (study B), 91 stray cats from a hotspot region in the northwest of Switzerland and 501 anaemic cats from across Switzerland (study C), and 65 Swiss domestic cats sampled in 2003 and 34 European wildcats from eastern France sampled in the period 1995–1996 (study D). The samples were analysed for Cytauxzoon spp. using real-time TaqMan quantitative PCR, and positive samples were subjected to 18S rRNA, cytochrome b ( CytB ) and cytochrome c oxidase subunit I ( COI ) gene sequencing. Results In study A, six of 13 cats from two neighbouring households in central Switzerland tested postive for Cytauxzoon spp.; two of the six infected cats died from bacterial infections. In studies B and C, only one of the 881 cats (0.1%; 95% confidence interval [CI]: 0–0.3%) in the countrywide survey and one of the 501 anaemic cats (0.2%; 95% CI: 0–0.6%) tested postive for Cytauxzoon spp. while eight of the 91 stray cats in the northwest of Switzerland tested positive (8.8%; 95% CI: 3.0–14.6%). In study D, Cytauxzoon spp. was detected in one of the 65 domestic cat samples from 2003 (1.5%; 95% CI: 0–4.5%) and in ten of the 34 European wildcat samples from 1995 to 1996 (29%; 95% CI: 14.2–44.7%). The isolates showed ≥ 98.6% sequence identities among the 18S rRNA, CytB and COI genes, respectively, and fell in the subclade Cytauxzoon europaeus based on CytB and COI gene phylogenetic analyses. Conclusions The study challenges the newly emerging nature of Cytauxzoon spp. in central Europe and confirms that isolates from domestic cats in Switzerland and European wild felids belong to the same species. Graphical Abstract

Cytauxzoon felis is a tick-transmitted intraerythrocytic apicomplexan infecting felids in the southeastern and midwestern United States. Bobcats (Lynx rufus) are the natural wildlife reservoir of C. felis, where in enzootic areas prevalence can reach 100%. Domestic cats can be subclinically infected with C. felis or can develop cytauxzoonosis. Two studies have documented the presence of C. felis in domestic cats in Illinois; these studies have shown a limited number of cases submitted to specialty labs. During 2014–2018, we obtained blood samples collected by veterinary staff from 514 cats that were apparently healthy and 74 cats that were suspected of cytauxzoonosis. These samples were screened using a sensitive, nested PCR to detect the presence of C. felis DNA. We herein document frequent occurrences of cytauxzoonosis (8–15 cases/year from 4 veterinary clinics) and 12.5% prevalence of subclinical infections in southern Illinois, a locality showing a sharp increase in cases of cytauxzoonosis. Our results suggest a high risk of cytauxzoonosis in southern Illinois, despite only recently being recognized in the area. We found no specific risk factors for cytauxzoonosis or subclinical infections in this location. In addition, cases of cytauxzoonosis occur every month of the year (with the highest frequency in summer) and therefore tick prevention should be used in domestic cats in enzootic regions throughout the year.

Cytauxzoon spp. have been detected in Brazil infecting mainly asymptomatic domestic cats and wild felids. However, the supposed genetic similarity with the pathogenic Cytauxzoon felis is questionable because it is based on analysis of short sequences of the 18S rRNA gene. Herein, we describe a novel Cytauxzoon species infecting an asymptomatic little-spotted-cat (Leopardus tigrinus) based on morphological, histopathological, and molecular analyses. The animal was attended presenting a history of a run-over with multiple traumas. Although the little-spotted-cat was stabilized, he died a few days later. Ring-shaped merozoites within erythrocytes were found on blood smears and in the abdominal effusion. In addition, schizonts were observed in histiocytes in the liver. Phylogenetic analyses based on both near-complete 18S rRNA and cytb genes positioned the obtained sequences in a unique clade, albeit closely related to Cytauxzoon felis from the USA. Genetic divergences ranging from 0.004 and 0.067–0.068 were found between the near-complete 18S rRNA and cytb sequences of Cytauxzoon sp. detected in the little-spotted-cat and C. felis, respectively. This study evidenced the circulation of a novel Cytauxzoon species, herein named Cytauxzoon brasiliensissp. nov., in an asymptomatic wild felid species from Brazil. Further studies are necessary to identify Cytauxzoon species from domestic and wild felids in the country.

Background Cytauxzoon felis is a life‐threatening protozoan disease of cats. Identification of schizont‐laden macrophages is a point‐of‐care diagnostic test for acute cytauxzoonosis. Hypothesis/Objectives The primary objective determined cytologic agreement between sample types to diagnose acute cytauxzoonosis. The secondary objective evaluated novices' ability to identify cytauxzoon organisms in blood films and tissue aspirates. Animals Thirty‐eight cats with suspected acute cytauxzoonosis and 5 controls examined postmortem. Methods Cases were prospectively submitted and collected. Blood film, lymph node, and splenic aspirates were blindly reviewed for sample quality, presence of schizont‐laden macrophages, and agreement between sample types. A subset of cases and controls were evaluated by 12 blinded novice observers to determine sensitivity and specificity for identifying organisms in various sample types. Results Acute cytauxzoonosis diagnosis was made on at least 1 sample type in 28/38 cats. Schizont‐laden macrophages were seen on 33% (10/30) of blood films, 56% (19/34) lymph node aspirates, 77% (26/34) splenic aspirates. Schizont‐laden macrophages were more likely seen on splenic than lymph node aspirates (McNemar's, P = .03) or blood film (McNemar's, P = <.001). Novice observers were more likely to agree with experts when identifying schizont‐laden macrophages in splenic aspirates (sensitivity = 77.1%, specificity = 94.4%) versus lymph node aspirates (sensitivity = 52.8%, specificity = 96.4%) or blood films (sensitivity = 41.7%, specificity = 96.9%). Conclusions and Clinical Importance Schizont‐laden macrophages are most frequently identified in spleen, even by novice observers. If the diagnosis of acute cytauxzoonosis cannot be confirmed via blood film, then splenic, followed by peripheral lymph node aspirates can be considered.

Urbanization alters components of natural ecosystems which can affect tick abundance and tick-borne disease prevalence. Likely due to these changes, tick-borne pathogen prevalence has increased in many U.S. urban areas. Despite the growing public health importance of tick-borne diseases, little is known about how they are influenced by urbanization in North America, especially in the central U.S. where several pathogens occur at or near their highest levels of incidence nationally. To determine whether urban development influences tick infection with bacteria and protozoa, we collected ticks at 16 parks across a gradient of urbanization intensity in Oklahoma City, Oklahoma, USA and tested them using a variety of PCR assays. Adult ticks tested positive for Rickettsia parkeri, R. amblyommatis, R. rhiphicephali, ‘Candidatus R. andeanae’, Ehrlichia chaffeensis, E. ewingii, Panola Mountain Ehrlichia, ‘Borrelia lonestari’, Theileria cervi, Babesia spp. Coco, and Cytauxzoon felis. These results indicate the presence of a high diversity of tick-borne bacteria and protozoa across an expanding urban area in the U.S. Great Plains. Although there appeared to be some risk of encountering tick-borne microorganisms across the entire urbanization gradient, E. chaffeensis, E. ewingii, and T. cerviinfected ticks and microbe diversity decreased with increasing urbanization intensity. We identified a low rate of coinfection between different microorganisms, with coinfected ticks mainly collected from sites in the least-urbanized areas. This study suggests the need for awareness of tick-borne disease risk throughout urban areas in the central U.S., and highlights a need for studies of tick host habitat use and movement in cities.

Background: Cytauxzoon felis is a protozoan parasite transmitted by ticks and affects felids. Acute infection in domestic cats is characterized by symptoms such as lethargy, anorexia, fever, and anemia. Methods: The present study focuses on diagnosing and molecularly identifying C. felis using a real-time PCR method in cats from Semnan, Iran. During the winter and spring of 2024, two hundred cats were randomly selected from veterinary clinics in Semnan. Blood samples were collected from the cats for DNA extraction and molecular analysis. Samples were divided into 40 pooled of 5 samples, each consisting of a combination of 5 blood samples. Then, the genomic DNAs were extracted from blood specimens and screened by real-time PCR (RT-PCR) for the presence of C. felis infection by amplifying of ITS2 gene belonging to the Cytauxzoon genus. Results: The results indicated that 6 out of the 200 blood samples were infected (3%). Conclusion: This study was conducted for the first time in Semnan and shows that the prevalence of C. felis in cats is significant.

Background Babesiosis is a protozoal, tick transmitted disease found worldwide in humans, wildlife and domesticated animals. Commonly used approaches to diagnose babesiosis include microscopic examination of peripheral blood smears, detection of circulating antibodies and PCR. To screen and differentiate canine Babesia infections many PCR assays amplify the 18S rRNA gene. These sequences contain hypervariable regions flanked by highly conserved regions allowing for amplification of a broad-range of Babesia spp. However, differences in the 18S rRNA gene sequence of distantly related clades can make it difficult to design assays that will amplify all Babesia species while excluding the amplification of other eukaryotes. By targeting Babesia mitochondrial genome (mtDNA), we designed a novel three primer qPCR with greater sensitivity and broader screening capabilities to diagnose and differentiate Babesia spp. Methods Using 13 Babesia mtDNA sequences, a region spanning two large subunit rRNA gene fragments ( lsu5 - lsu4 ) was aligned to design three primers for use in a qPCR assay (LSU qPCR) capable of amplifying a wide range of Babesia spp. Plasmid clones were generated and used as standards to determine efficiency, linear dynamic range and analytical sensitivity. Animals naturally infected with vector-borne pathogens were tested retrospectively and prospectively to determine relative clinical sensitivity and specificity by comparing the LSU qPCR to an established 18S rDNA qPCR. Results The LSU qPCR efficiencies ranged between 92 and 100% with the limit of detection at five copies/reaction. The assay did not amplify mammalian host or other vector-borne pathogen gDNA except Cytauxzoon felis (a feline protozoal pathogen). The LSU qPCR assay amplified 12 different Babesia . sp. and C. felis from 31/31 (100%) archived samples, whereas the 18S qPCR amplified only 26/31 (83.9%). By prospective analysis, 19/394 diagnostic accessions (4.8%) were LSU qPCR positive, compared to 11/394 (2.8%) 18S rDNA qPCR positive. Conclusions We have developed a more sensitive qPCR assay with a more expansive range of Babesia spp. detection by targeting a highly conserved region of mtDNA, when compared to an established 18S qPCR.

Objectives The aim of this study was to evaluate the blood of cats in Colorado, USA, with suspected infectious causes of anemia for the presence of Babesia species and Cytauxzoon felis DNA. Results of PCR testing for other common vector-borne diseases potentially associated with anemia are also reported. Methods Samples from 101 cats were tested using a PCR assay that coamplified the DNA of C felis and Babesia species mitochondrial DNA. PCR testing for DNA of hemoplasmas, Bartonella species, Ehrlichia species, Anaplasma species, Neorickettsia risticii and Wolbachia genera was also performed if not carried out previously. Results Twenty-two cats (21.8%) were positive for DNA of an infectious agent. DNA from hemoplasma species were amplified from 14 cats (13.9%). Bartonella species DNA was amplified from four cats (4%) and Ehrlichia canis, Anaplasma platys, Anaplasma phagocytophilum and Wolbachia genera DNA were amplified from one cat each. Babesia species and C felis mitochondrial DNA were not amplified from any sample. Conclusions and relevance Based on the results of this study, it does not appear that Babesia species or C felis are clinically relevant in anemic cats in Colorado, USA. For C felis, this suggests that the vector Amblyomma americanum is still uncommon in this geographic area.

Domestic and wild felids are frequently parasitized by apicomplexan protozoa in the genus Cytauxzoon. Expanding species diversity has recently been described within this genus, with potential implications for epidemiology and pathogenesis. In light of these findings, this study assessed the genetic diversity of Cytauxzoon spp. in wild felids (n = 66) from different eco-regions of Brazil and Argentina. Of the 66 blood samples analyzed, 53 (80.3%) were 18S rRNA gene PCR-positive for Cytauxzoon spp., including 43 jaguars (Panthera onca) and 10 ocelots (Leopardus pardalis). Panthera onca specimens (100%, 43/43) were most frequently infected, followed by Leopardus pardalis (76.9%; 10/13). Cytauxzoon spp. were not detected in Leopardus braccatus (n = 1) or Puma concolor (n = 9). Phylogenetic analyses of fragments of the 18S rRNA, cytB, and cox-1 gene sequences from jaguars were closely related to Cytauxzoon felis. In contrast, sequences from ocelots were more closely associated with Cytauxzoon brasiliensis. Distance and haplotype analysis further confirmed the circulation of at least two distinct genovariants of C. felis among jaguars, as evidenced by their close positioning and low genetic divergence (0–0.14% for 18S rRNA, 0.37–0.56% for cytB, and 0.08–0.74% for cox-1). Additionally, sequence data from ocelots suggested that multiple genovariants of C. brasiliensis are circulating among these cats in different Brazilian eco-regions. Our study provides evidence of two distinct Cytauxzoon organisms parasitizing free-ranging and captive jaguars and ocelots, respectively, in Brazil and Argentina.