One Health Approach to Rickettsiosis: A Five-Year Study on Spotted Fever Group Rickettsiae in Ticks Collected from Humans, Animals and Environment

The spotted fever group of Rickettsiae is a heterogeneous group of Rickettsiae transmitted by ticks, causing similar diseases in humans (spotted fever). Until recently, it was supposed that a single pathogenic tick-borne SFG Rickettsia circulated in each different geographic area and that R. conorii subsp. conorii was the SFG Rickettsiae circulating in Italy, but in the last decade, thanks to molecular diagnostic, several different Rickettsia species, previously not considered pathogenic for decades, have been isolated from ticks and definitively associated to human disease, also in Italy. The present survey was carried out with the aim of investigating the presence of different SFG Rickettsia species in a geographic area where no information was available. Ticks collected from animals submitted to necropsy, removed from humans in local hospitals and collected from the environment were identified and tested by PCR for Rickettsia spp. based on the gltA gene, and positive PCR products were sequenced. A total of 3286 ticks were collected. Fifteen tick species were recognized, the most represented (79.52%) species in the collection was Ixodes ricinus, followed by Rhipicephalus sanguineus (9.13%). The overall prevalence of Rickettsia infection was 7.58%. Eight species of Rickettsia were identified, the most frequent was R. monacensis (56%), followed by R. helvetica (25.50%). Noteworthy, is the detection in the present study of Rrhipicephali, detected only twice in Italy. These are the first data available on SFG Rickettsiae circulation in the study area and they can be considered as starting point to assess the possible risk for humans.

Melting curve analysis to differentiate Rickettsia spp. and Rickettsia felis

Rickettsiosis, caused by Rickettsia species, is one of the old arthropod-borne illness that commonly found in humans and animals. One of the barriers to rickettsiosis control is the intricacy and time-consuming nature of rickettsiosis laboratory diagnosis. This study aimed to establish quantitative real-time PCR targeting the gltA gene for the DNA differentiation of Rickettsia spp. and Ricketsia felis. The collection of cat flea was extracted to acquire the DNA of Rickettsia. Primers were designed based on the analysis of Rickettsia gltA gene sequences. The confirmation of R. felis was performed by sequencing of PCR product. BLAST analysis was done to confirm the closest similarity of the sequences. Results of this study highlighted the melting temperature was reached at 78,5 °C for Rickettsia spp. and 76.5+0.5 °C for Rickettsia felis. The melting peak temperatures were significantly different between Rickettsia spp. and R. felis (p<0.05). The findings of this work are crucial in the development of powerful diagnostic procedures for detecting and distinguishing Rickettsia spp. and R. felis species.

273. Neurological Involvement Caused by Intracellular Bacteria

Background Infection of the central nervous system is a severe and fatal disease. Causative agents include bacteria, viruses or fungi. Intracellular bacteria are not only overlooked, but also underdiagnosed. We aimed to study the clinical, laboratory and evolutionary features of neurological involvement caused by intracellular bacteria. Methods We conducted a retrospective study including all patients hospitalized in the infectious disease department for neurological involvement caused by intracellular bacteria between 1995 and 2020. The diagnosis was confirmed by serology. Results We encountered 76 cases among which 43 were males (56.6%). The mean age was 32±18 years. The revealing symptoms included fever (97.4%), cephalalgia (73.7%), vomiting (64.5%) and arthralgia (51.3%). Lumbar puncture revealed a median white blood cell count of 120[56-340]/mm3. Lymphocytic pleocytosis was noted in 62% of the cases. Elevated cerebrospinal fluid (CSF) protein level was noted in 37 cases (48.7%) with a median of 0.84[0.6-1.37] g/L. Low CSF fluid glucose level was noted in 14 cases (18.4%). There were 70 cases (92.1%) of meningitis and 6 cases of meningoencephalitis (7.9%). The causative agent included Rickettsia species in 47 cases (61.8%), Brucella species in 17 cases (22.4%) and Mycoplasma species in 12 cases (15.8%). Laboratory investigations included elevated C-reactive protein levels (40.7%), thrombocytopenia (32.8%) and increase in hepatic enzyme levels (21%). Anemia was noted in 27 cases (35.5%), leukocytosis in 24 cases (31.5%) and leucopoenia in 6 cases (7.8%). Blood and CSF cultures were positive for Brucella in 2 cases (2.6%) and 5 cases (6.5%), respectively. The mean duration of treatment was 156±94 days for brucellosis cases, 9±4 days for rickettsiosis cases and 10±6 days for Mycoplasma cases. The disease evolution was favorable in 72 cases (94.7%). Four patients were dead (5.3%). Complications were noted in 5 cases (6.5%) and sequelae in 2 cases (2.6%). Conclusion Intracellular bacteria including Brucella, Rickettsia and Mycoplasma species should be considered in front of neurological symptoms. Meningitis with lymphocytic pleocytosis was the most common clinical presentation. An early diagnosis followed by the adequate treatment might avoid complications and death. Disclosures All Authors: No reported disclosures

An Update on the Laboratory Diagnosis of Rickettsia spp. Infection

Rickettsia species causing human illness are present globally and can cause significant disease. Diagnosis and identification of this intracellular bacteria are challenging with many available diagnostic modalities suffering from several shortcomings. Detection of antibodies directed against Rickettsia spp. via serological methods remains widely used with a broad range of sensitivity and specificity values reported depending on the assay. Molecular methods, including polymerase chain reaction (PCR) testing, enables species-specific identification with a fast turnaround time; however, due to resource requirements, use in some endemic settings is limited. Reports on the use of next-generation sequencing (NGS) and metagenomics to diagnose Rickettsia spp. infection have been increasing. Despite offering several potential advantages in the diagnosis and surveillance of disease, genomic approaches are currently only limited to reference and research laboratories. Continued development of Rickettsia spp. diagnostics is required to improve disease detection and epidemiological surveillance, and to better understand transmission dynamics.

Rickettsia lusitaniae in Ornithodoros Porcinus Ticks, Zambia

Rickettsial pathogens are amongst the emerging and re-emerging vector-borne zoonoses of public health importance. Though traditionally considered to be transmitted by ixodid ticks, the role of argasid ticks as vectors of these pathogens is increasingly being recognized. While bat-feeding (Ornithodoros faini) and chicken-feeding (Argas walkerae) argasid ticks have been shown to harbor Rickettsia pathogens in Zambia, there are currently no reports of Rickettsia infection in southern Africa from warthog-feeding (Phacochoerus africanus) soft ticks, particularly Ornithodoros moubata and Ornithodoros porcinus. Our study sought to expand on the existing knowledge on the role of soft ticks in the epidemiology of Rickettsia species through screening for Rickettsia pathogens in warthog burrow-dwelling soft ticks from two national parks in Zambia. The tick species from which Rickettsia were detected in this study were identified as Ornithodoros porcinus, and an overall minimal Rickettsia infection rate of 19.8% (32/162) was observed. All of the sequenced Rickettsia were identified as Rickettsia lusitaniae based on nucleotide sequence similarity and phylogenetic analysis of the citrate synthase (gltA) and 17kDa common antigen (htrA) genes. Utilizing all of the gltA (n = 10) and htrA (n = 12) nucleotide sequences obtained in this study, BLAST analysis showed 100% nucleotide similarity to Rickettsia lusitaniae. Phylogenetic analysis revealed that all of the Zambian gltA and htrA gene sequences could be grouped with those of Rickettsia lusitaniae obtained in various parts of the world. Our data suggest that Rickettsia lusitaniae has a wider geographic and vector range, enhancing to our understanding of Rickettsia lusitaniae epidemiology in sub-Saharan Africa.

Prevalence of Vector-Borne Pathogens in Reproductive and Non-Reproductive Tissue Samples from Free-Roaming Domestic Cats in the South Atlantic USA

Reservoir to multiple species of zoonotic pathogens, free-roaming cats (FRCs) interact with domestic and wild animals, vectors, and humans. To assess the potential for feline vector-borne pathogens to be vertically transmitted, this study surveyed ear tip and reproductive tissues of FRCs from two locations in the South Atlantic United States for Anaplasma, Bartonella, Ehrlichia, hemotropic Mycoplasma, and Rickettsia species. We collected ovary (n = 72), uterus (n = 54), testicle (n = 74), and ear tip (n = 73) tissue from 73 cats, and fetal (n = 20) and placental (n = 19) tissue from 11 queens. Pathogen DNA was amplified utilizing qPCR, confirmed by sequencing. Cats were more frequently Bartonella henselae positive on reproductive tissues (19%, 14/73) than ear tip (5%, 4/73; p = 0.02). B. henselae was amplified from fetus (20%, 4/20) and placenta samples (11%, 2/19). Bartonella spp. infection was more common in cats from North Carolina (76%, 26/34) than Virginia (13%, 5/39; p < 0.0001). Fourteen percent (10/73) of both ear tip and reproductive tissues were positive for hemotropic Mycoplasma spp. Anaplasma, Ehrlichia, and Rickettsia spp. DNA was not amplified from any cat/tissue. These findings suggest that B. henselae preferentially infected cats’ reproductive tissue and reinforces the importance of investigating the potential for B. henselae vertical transmission or induction of reproductive failure.

CLINICAL AND LABORATORY CHARACTERISTICS OF TICK-BORNED RICKETTSIOSIS RELATED TO RICKETTSIA SIBIRICA AND "CANDIDATUS RICKETTSIA TARASEVICHIAE"

The Siberian tick-borne typhus (STT) is the most common tick-borne rickettsiosis (TBR) in Russia, registered in 17 administrative territories of the Southern Siberia and the Far East. The aim of this study was to describe clinical picture, pathological data and the results of laboratory diagnostics during fatal mixed infection caused by two rickettsia species most common in Russia - pathogenic Rickettsia sibirica and poorly examined Candidatus R. tarasevichiae, including identification of its etiological agents.A four-year-old girl in the Krasnoyarsk Territory in a hyperendemic focus of the Siberian tick-borne typhus after tick sucking revealed typical TBR symptoms (scab at the site of tick suction, fever, spotted rash, myalgia) and meningeal syndrome, which is not typical for the Siberian tick-borne typhus. The child died on the seventh day of the illness. Autopsy data (hepatosplenomegaly; cerebral edema, which was the immediate cause of death) and the results of histological examination (productive vasculitis of the brain, spinal cord and skin, polymorphic cell perivascular infiltrates in the liver and lungs, serous meningitis, myeloid hyperplasia of the spleen and lymph nodes, interstitial lymphoid infiltration in the myocardium) confirmed the clinical diagnosis of tick-borne rickettsiosis.The patient's blood and brain samples were tested for a wide range of tick-borne pathogens and enteric viruses that cause brain damage using PCR followed by sequencing of the positive samples.The DNA of Rickettsia sibirica and "Candidatus Rickettsia tarasevichiae" was found in both blood and brain samples. R. sibirica was identified by the nucleotide sequences of gene fragments gltA, OmpA and ompB, and "Candidatus R. tarasevichiae" - the gltA and ompB genes using nested PCR and sequencing. All amplified fragments were sequenced in both directions; the obtained sequences were deposited in the GenBank database under the inventory numbers: MK048467-MK048475. We have not identified other tick-borne pathogens or intestinal viruses in the patient samples able to result in meningeal syndrome.The area where the child was sucked by the tick belongs to the regions with a high incidence of STT. Several species of mites coexist in this area; of these, Haemaphisalis concinna, the carrier of R. sibirica, dominates the populations of Ixodes mites, while Ixodes persulcatus, the main reservoir of "Candidatus R. tarasevichiae", is less common.As a result of the conducted studies, for the first time in the Russian Federation, a verified case of a lethal infection associated with two species of rickettsiae - Rickettsia sibirica and "Candidatus R. tarasevichiae" was identified and described.

Molecular Detection and Genetic Identification of Rickettsia Infection in Ixodes granulatus Ticks, an Incriminated Vector for Geographical Transmission in Taiwan

Tick-borne Rickettsia pathogens have become an emerging source of zoonotic infections and have a major impact on human health worldwide. In this study, the prevalence and genetic identity of Rickettsia infections in Ixodes granulatus ticks was firstly determined in Kinmen Island of Taiwan. A total of 247 I. granulatus ticks were examined for Rickettsia infection by nested-PCR assay targeting the citrate synthase (gltA) gene of Rickettsia. The Rickettsia infection was detected with a general infection rate of 4.86%, and was detected in nymph, male and female stages with an infection rate of 3.81%, 0% and 6.84%, respectively. Phylogenetic relationships were analyzed by comparing the gltA sequences obtained from four Taiwan strains and 19 other strains representing 13 genospecies of Rickettsia. Phylogenetic analyses reveal that all Taiwan strains were genetically affiliated to the genospecies of spotted fever (R. parkeri) and transitional (R. felis) groups of Rickettsia. Our findings reveal the first detection of R. parkeri-like and R. felis in I. granulatus ticks from Kinmen Island. As a tourist island between Taiwan and mainland China, these results demonstrate the epidemiological significance of diverse Rickettsia species existed in I. granulatus ticks and highlight the potential threat of geographical transmission among humans in the Taiwan area.