17-kDa Antigen Research Articles

Estimating the Seroincidence of Scrub Typhus using Antibody Dynamics after Infection.

Scrub typhus, a vector-borne bacterial infection, is an important but neglected disease globally. Accurately characterizing the burden is challenging because of nonspecific symptoms and limited diagnostics. Prior seroepidemiology studies have struggled to find consensus cutoffs that permit comparisons of estimates across contexts and time. In this study, we present a novel approach that does not require a cutoff and instead uses information about antibody kinetics after infection to estimate seroincidence. We use data from three cohorts of scrub typhus patients in Chiang Rai, Thailand, and Vellore, India, to characterize antibody kinetics after infection and two population serosurveys in the Kathmandu Valley, Nepal, and Tamil Nadu, India, to estimate seroincidence. The samples were tested for IgM and IgG responses to Orientia tsutsugamushi-derived recombinant 56-kDa antigen using commercial enzyme-linked immunosorbent assay kits. We used Bayesian hierarchical models to characterize antibody responses after scrub typhus infection and used the joint distributions of the peak antibody titers and decay rates to estimate population-level incidence rates in the cross-sectional serosurveys. Median responses persisted above an optical density (OD) of 1.8 for 23.6 months for IgG and an OD of 1 for 4.5 months for IgM. Among 18- to 29-year-olds, the seroincidence was 10 per 1,000 person-years (95% CI, 5-19) in Tamil Nadu, India, and 14 per 1,000 person-years (95% CI: 10-20) in the Kathmandu Valley, Nepal. When seroincidence was calculated with antibody decay ignored, the disease burden was underestimated by more than 50%. The approach can be deployed prospectively, coupled with existing serosurveys, or leverage banked samples to efficiently generate scrub typhus seroincidence estimates.

Flea-Borne Rickettsioses and Scrub Typhus in Patients with Suspected Arbovirus Infection in Bangkok, Thailand.

Background: In urban Thailand, arboviral infections dominate diagnoses of acute undifferentiated fevers (AUFs) owing to their well-defined epidemiology and characteristic clinical presentations. However, rickettsial diseases, also endemic in this setting, remain under-recognized owing to challenges in early detection. Objective: This study aimed to identify potential rickettsial infections among patients with AUF in Bangkok and vicinity utilizing leftover nucleic acid extracted from serum samples from patients initially suspected of but negative for arbovirus infections. Materials and Methods: A total of 609 nucleic acid samples were screened for rickettsial bacteria using real-time PCR, targeting the 17-kDa common antigen gene of Rickettsia spp. and the 47-kDa gene of Orientia tsutsugamushi. Results: Nine samples were positive for Rickettsia spp. and two were positive for O. tsutsugamushi. DNA sequence and phylogenetic analyses based on partial 17-kDa antigen and citrate synthase (gltA) genes identified the Rickettsia-positive samples as R. typhi in eight cases and R. felis in one case. Analysis of the 56-kDa type-specific antigen gene identified the two O. tsutsugamushi isolates as Gilliam-related genotypes. Although rickettsial diseases typically present with mild symptoms, two patients with R. typhi infection (murine typhus) developed respiratory distress syndrome, highlighting the potential for rare but serious complications. Conclusion: This study underscores the critical importance of differential diagnosis and prompt, effective intervention to prevent complications in suspected cases.

Pathological Responses in Asian House Shrews (Suncus murinus) to the Naturally Acquired Orientia tsutsugamushi Infection.

Scrub typhus is a re-emerging disease caused by Orientia tsutsugamushi, transmitted by mites belonging to the family Trombiculidae. Humans and rodents acquire the infection by the bite of larval mites/chiggers. Suncus murinus, the Asian house shrew, has been reported to harbor the vector mites and has been naturally infected with O. tsutsugamushi. The present study aimed to localize and record O. tsutsugamushi in the tissues and the host response in shrews naturally infected with O. tsutsugamushi. Sheehan's modified May-Grunwald Giemsa staining was carried out in 365 tissues from 87 animals, and rickettsiae were documented in 87 tissues from 20 animals. Immunohistochemical (IHC) staining, using polyclonal antibodies raised against selected epitopes of the 56-kDa antigen, was carried out, and 81/87 tissue sections were tested positive for O. tsutsugamushi. By IHC, in addition to the endothelium, the pathogen was also demonstrated by IHC in cardiomyocytes, the bronchiolar epithelium, stroma of the lungs, hepatocytes, the bile duct epithelium, the epithelium and goblet cells of intestine, the tubular epithelium of the kidney, and splenic macrophages. Furthermore, the pathogen was confirmed by real-time PCR using blood (n = 20) and tissues (n = 81) of the IHC-positive animals. None of the blood samples and only 22 out of 81 IHC-positive tissues were tested positive by PCR. By nucleotide sequencing of the 56-kDa gene, Gilliam and Karp strains were found circulating among these animals. Although these bacterial strains are highly virulent and cause a wide range of pathological alterations, hence exploring their adaptive mechanisms of survival in shrews will be of significance. Given that the pathogen localizes in various organs following a transient bacteremia, we recommend the inclusion of tissues from the heart, lung, intestine, and kidney of reservoir animals, in addition to blood samples, for future molecular surveillance of scrub typhus.

Diagnosis of Rickettsial infection by Multiplex Real-time PCR in Febrile Pediatric Patients Admitted in a Tertiary Care Hospital at Chennai, South India

Rickettsioses caused by Rickettsia and Orientia spp. are the re-emerging diseases in India, which are grossly underdiagnosed, particularly among children. They usually present as mild-febrile illness but may extend to severe life-threatening complications. Early diagnosis followed by proper treatment reduces the morbidity and mortality. Non-specific clinical symptoms and lack of point of care diagnosis may delay the treatment. Molecular assays like PCR may helpful in the early diagnosis and confirmation of rickettsial diseases. In this study, we used multiplex real-time PCR to detect Rickettsia spp. and Orientia spp. in febrile pediatric patients. Whole blood was collected from 239 clinically suspected febrile pediatric patients aged between 6 months to 12 years admitted in tertiary care hospital at Chennai, South India. Multiplex real-time PCR was used to target the gltA gene for Rickettsia spp. and the 47kDa gene for Orientia tsutsugamushi. To compare the sensitivity, nested PCR was performed on the 56kDa antigen gene of O. tsutsugamushi and the Rickettsia genus specific gltA gene. By multiplex real-time PCR, 15 samples were positive for O. tsutsugamushi and 3 were positive for Rickettsia spp. Nested PCR identified 35 positive samples for O. tsutsugamushi and 4 positive samples for Rickettsia spp. Even though multiplex real-time PCR had lower positivity than nPCR, it was effective in diagnosing O. tsutsugamushi and Rickettsia spp. in a single assay.

Distribution of ticks and their possession of spotted fever group Rickettsia in Ibaraki prefecture

IntroductionTick-borne diseases (TBDs) are a growing threat in Japan. However, distribution of ticks and their possession of human pathogens remain poorly understood. MethodsIn the present study, we collected 3477 ticks at 6 remote, woodland sites in Ibaraki prefecture between May 23 and November 4, 2021, and investigated the distribution and the possession of spotted fever group Rickettia (SFGR). ResultsThe collected ticks included Haemaphysalis flava (78.3 %), Haemaphysalis longicornis (9.0 %), Haemaphysalis hystricis (4.6 %), Ixodes turdus (4.3 %), Amblyomma testudinarium (2.1 %), Haemaphysalis cornigera (0.9 %), Haemaphysalis formosensis (0.9 %), Haemaphysalis megaspinosa (0.2 %), Ixodes ovatus (0.1 %), Ixodes nipponensis (0.09 %), and Ixodes columnae (0.03 %). Of 2160 DNA samples extracted from the ticks, the gltA gene and the 17-kDa antigen gene of SFGR were detected in 67 samples. Among 1682 samples from adult and nymph ticks, the positive rate of SFGR was 2.7 %. Sequence analyses of the partial 17-kDa antigen gene demonstrated that the detected SFGR were classified into 8 groups (G1 to G8). The sequences of G2, G4, G5, G6, and G7 were either identical to or differed by one base pair from those of Rickettsia asiatica, Rickettsia tamurae, Rickettsia monacensis, Rickettsia canadensis, and Rickettsia felis, respectively. ConclusionThe present study revealed a diverse tick fauna in Ibaraki prefecture, including detection of species commonly found in southwestern Japan. Although the prevalence of SFGR in ticks was lower than in previous studies, several SFGR causing human infection may be present.

Phylogenetic Analysis of Spotted Fever Group Rickettsia Gene from Ticks and Human Patients in Tottori Prefecture, Japan.

Japanese spotted fever (JSF) is a tick-borne bacterial febrile disease caused by Rickettsia japonica characterized by fever, rash, and occasional death. The number of patients in Japan and the Tottori Prefecture has been increasing over the past 20 years. Most cases were found in Eastern Tottori; however, the distribution of patients has expanded to the Central and Western regions. Ticks carried by wild animals may be the cause, but the prevalence of R. japonica in ticks has not yet been analyzed. Ticks were collected by flagging-dragging from 16 sites in Tottori, Japan. The ticks were morphologically classified and DNA was extracted. The 17-kDa antigen gene was amplified using nested PCR. PCR amplicons from ticks and JSF patients were sequenced and phylogenetically compared. In total, 177 ticks were collected and identified as Haemahysalis, Ixodes, Amblyomma, and Dermcentor. The Spotted Fever Group Rickettsia (SFGR) was detected in Haemahysalis and Amblyomma spp. using PCR, with positivity rates of 36.8% and 33.3%, respectively. DNA sequencing and phylogenetic analysis revealed that positive ticks harbored R. japonica, P. raoultii, and other Rickettsiae species; however, the patient's samples were restricted to R. japonica. Similar to the incidence of JSF, the rate of R. japonica-positive ticks was higher in the Eastern region; however, R. japonica-positive ticks were also detected in the Western region. R. japonica sequences had been found in ticks collected in Tottori Prefecture. Ticks harboring R. japonica were found in the Eastern and Western parts of Tottori Prefecture and the sequences were identical to the human cases. Only the R. japonica sequence has been detected in patients with spotted fever symptoms, even though ticks were harboring various SFGRs.

Orientia tsutsugamushi: Diversity and Distribution in India: A Review

Scrub typhus is a disease caused by Orientia tsutsugamushi which is an obligate, intracellular gram-negative bacterium. It is endemic to a part of the world known as “tsutsugamushi triangle.” This extends from Japan, China, Taiwan, South Koria, Nepal, Pakistan, Papua New Guinea, Australian states of Queensland and Northern New South Wales. In India, it has been recognized for several years and the disease widely spread all over the country. The severity of scrub typhus disease has been reported to be strain dependent in humans. Orientia tsutsugamushi strains have been examined by various research tools. PCR amplification and sequencing of 56-kDa antigen gene is one of the most advanced tool in identifying O. tsutsugamushi strains and genotypes. In the present review, English-language literature for reports of Orientia tsutsugamushi genotype diversity and distribution in India was searched using PubMed and Google Scholar databases. The search terms scrub typhus or mite typhus or tropical typhus or tsutsugamushi disease or Orientia tsutsugamushi or Rickettsia tsutsugamushiwere used in combination with the term genotypes or strains or serotypes. The result of present review revealed limited Information related to genotype diversity and distribution in the country. Understanding a more detailed regional genetic diversity is required in region-specific vaccine development strategies andsero-diagnostics tools.

Detection of Bartonella spp. and Rickettsia spp. in cat fleas (Ctenocephalides felis) collected from free-roaming domestic cats in southeastern Georgia, USA.

The cat flea (Ctenocephalides felis) is a competent vector of numerous bacterial pathogens in the genera Bartonella and Rickettsia. In the United States, free-roaming domestic cats (Felis catus) increase the risk of exposure to C. felis for humans and their companion animals. In collaboration with a trap-neuter-return program, we collected fleas from 283 feral/stray cats in southeastern Georgia between May and July of 2020. A total of 3,643 flea specimens were collected, and C. felis was the only flea species recovered from all cats sampled. The mean number of fleas per cat host was highest in the month of June when compared to May and July, and higher in juvenile cats (< 1year) than the adults (≥ 1year). Real-time PCR assays were used to test a subset of the collected fleas (n=468) for the presence of Bartonella spp. and Rickettsia spp. DNA. Among those flea pools tested, 35.2% were positive for genus-specific citrate synthase gene of Bartonella, 16.5% were positive for the genus-specific 17-kDa protein antigen gene of Rickettsia, and none were positive for the species-specific outer membrane protein B gene of Rickettsia typhi. The identification of potential flea-borne pathogens was more frequent from the subset of C. felis collected in May, and female cats had more Bartonella-positive fleas and less Rickettsia-positive fleas than male cats. Overall, the present study provides valuable insights into the frequency of C. felis from outdoor community cats in southeastern Georgia, and highlights the possible risk for human exposure to potential flea-borne pathogens.

A molecular survey of spotted fever group rickettsiae in introduced raccoons (Procyon lotor)

BackgroundThe raccoon Procyon lotor (Linnaeus, 1758) (Carnivora; Procyonidae) is one of the most important and most intensively studied invasive mammal species in Europe. Within the last 30 years the raccoon has spread at an increasing rate, resulting in the establishment of local populations in various regions of Europe. In these newly colonised areas, gaps in knowledge of the raccoon’s biology concern not only most aspects of its ecology in a broad sense, but also its pathogens and parasites. Most micropathogens recorded hitherto in the raccoons that have colonised Europe have documented epizootic and zoonotic potential. Thus, it is considered especially important to investigate the role played by the raccoon in the spread of pathogens through both animal-animal and animal-human pathways.MethodsTissue samples of raccoons from Poland and Germany were examined in this study. In total, 384 tissue samples from 220 raccoons (170 spleen samples, 82 liver biopsies, 132 ear biopsies) were examined using molecular methods. The presence of Rickettsia spp. DNA was screened through amplification of a fragment of the gltA gene. Samples that were PCR positive for gltA were tested for other rickettsial genes, ompB and a 17-kDa antigen. For taxonomic purposes, the obtained sequences were compared with corresponding sequences deposited in GenBank using the Basic Local Alignment Search Tool, and phylogenetic analyses were conducted using Bayesian inference implemented in MrBayes software.ResultsRickettsia DNA was confirmed only in skin biopsies; no isolates from the spleen or liver were positive for Rickettsia DNA. With the exception of one sample from Germany, which was positive for Rickettsia helvetica DNA, all the samples positive for Rickettsia DNA derived from the Polish population of raccoons. DNA of Rickettsia spp. was detected in 25 samples, i.e. 11.4% of the tested raccoons, and R. helvetica was confirmed in 52% of the positive samples. Additionally, single cases of Rickettsia monacensis, Rickettsia raoultii, and Candidatus Rickettsia kotlanii-like were found, and in 32% of all the positive samples similarity was shown to different Rickettsia endosymbionts. Out of the samples that tested positive for gltA, amplicons of ompB and 17 kDa were successfully sequenced from 14 and three samples, respectively.ConclusionsTo the best of our knowledge, this study provides, for the first time, evidence of the occurrence of Rickettsia pathogens and endosymbionts in the European population of raccoons. Further, broader research on different species of wild vertebrates, and ticks, as potential vectors and hosts for tick-borne pathogens, in natural as well as in peri-urban environments, is therefore required.Graphical abstract

Demographic and Clinical Characteristics of Scrub Typhus Cases detected by Nested Polymerase Chain Reaction among Febrile Patients in Mymensingh, Bangladesh

Background &amp; objectives: Scrub typhus is a mite borne rickettsial illness, caused by the bacterium Orientia tsutsugamushi, which is endemic to Asia pacific region. Due to wide spectrum of non-specific manifestations and inaccessibility of accurate diagnostic tools, it remains underdiagnosed in many countries of Southeast Asia including Bangladesh, resulting in severe life-threatening complications. Therefore, this study was aimed to diagnose scrub typhus through molecular detection of O. tsutsugamushi by ‘Nested Polymerase Chain Reaction (N-PCR)’ among febrile patients in Mymensingh, Bangladesh &amp; to study the socio-demographic as well as clinical characteristics of the diagnosed cases. Methods: It was a cross-sectional type of study, conducted at department of Microbiology, Mymensingh Medical College between March 2018 and February 2019. Blood samples were taken from 453 febrile patients of suspected rickettsial illness, referred from both inpatient and outpatient facility of Department of Medicine and Department of Pediatrics, Mymensingh Medical college Hospital (MMCH). Then DNA was extracted from the whole blood, following Phenol-Chloroform extraction &amp; ethanol precipitation method. Finally, N-PCR was performed on extracted DNA, targeting 47Kda antigen gene, to detect O. tsutsugamushi. Results: Out of 453, this study documented [78 (17.21%)] PCR positive scrub typhus cases, among which majority [42 (53.84%)] were female &amp; maximum [26 (33.33%)] were in age group &gt;15-30 years. Higher number of PCR positive patients [51 (65.39%)] were from rural areas. Among the scrub typhus cases, myalgia (48; 61.53%) was the most common manifestation, followed by headache (44; 56.41%) and cough (44; 56.41%). Eschar was present only in 14 (17.94%) cases and 8 (10.25 %) patients had skin rashes. Leukocytosis and leukopenia were documented in 13 (16.66%) and 5 (6.41%) cases respectively. Decreased Hb% was recorded in 14(17.9%) &amp; 16 (20.5%) cases had thrombocytopenia. Conclusion: This study recorded a high number of scrub typhus cases in Mymensingh region with non-specific febrile manifestations, hindering early &amp; accurate clinical diagnosis. Complications like - renal &amp; hepatic impairment, CNS involvement, ARDS as well as hematological abnormalities were documented also. So, scrub typhus should be considered in the differential diagnosis of undifferentiated febrile illness in Bangladesh. Further molecular studies on scrub typhus might be done throughout the countries to create awareness among physicians &amp; to evaluate actual disease burden. Mediscope 2022;9(1): .30-40

Molecular Characterization by Multilocus Sequence Typing and Diversity Analysis of Rickettsia asembonensis in Peru.

Despite several reports worldwide documenting the presence of Rickettsia asembonensis in samples derived from ectoparasites, animals and more recently humans, genomic information of these specimens remains scarce, and when available, is usually limited to small genomic fragments of limited value. We generated complete sequences for two conserved (17-kDa antigen gene and gltA) and three variable (sca4, ompB and ompA) genes in five R. asembonensis DNA samples detected in cat and dog fleas in Peru. Complete gene sequences were used to conduct multi-locus sequence typing and phylogenetic analyses to assess diversity and infer relationships among strains and other reference sequences. The 17-kDa antigen gene was highly conserved across Rickettsia species. Of the variable genes ompB was the most variable, but this diversity was not captured through phylogenetics alone even when efforts were made to maximize potential diversity in terms of flea species, animal host and location. Through a combination of de novo and reference-based genome assembly we identified a 75 bp insertion in ompA that encodes a 25 aa repetitive motif found in other Rickettsia species, but not present in the original prototype strain from Kenya. R. asembonensis has only recently been shown to be a bona-fide human pathogen. As such, and compounded by a lack of available genomic information, it remains understudied. Our work directly addresses the lack of genomic information available worldwide for the study of these novel Rickettsia species and specifically contributes to our understanding of the diversity and molecular epidemiology of R. asembonensis in Peru.

Islet Autoimmunity is Highly Prevalent and Associated With Diminished β-Cell Function in Patients With Type 2 Diabetes in the Grade Study.

Islet autoimmunity may contribute to β-cell dysfunction in type 2 diabetes (T2D). Its prevalence and clinical significance have not been rigorously determined. In this ancillary study to the Glycemia Reduction Approaches in Diabetes-A Comparative Effectiveness (GRADE) Study, we investigated the prevalence of cellular and humoral islet autoimmunity in patients with T2D duration 4·0±3·0 y, HbA1c 7·5±0·5% on metformin alone. We measured T cell autoreactivity against islet proteins, islet autoantibodies against GAD65, IA2, ZnT8, and β-cell function. Cellular islet autoimmunity was present in 41·3%, humoral islet autoimmunity in 13·5%, and both in 5·3%. β-cell function calculated as iAUC-CG and ΔC-peptide(0- 30)/Δglucose(0-30) from an oral glucose tolerance test was lower among T cell-positives (T+) than T cell-negatives (T-) using two different adjustments for insulin sensitivity (iAUC-CG: 13·2% [95% CI 0·3, 24·4%] or 11·4% [95% CI 0·4, 21·2%] lower; ΔC-peptide(0-30)/Δglucose(0-30)) 19% [95% CI 3·1, 32·3%] or 17·7% [95% CI 2·6, 30·5%] lower). T+ patients had 17% higher HbA1c (95% CI 0·07, 0·28) and 7·7 mg/dL higher fasting plasma glucose levels (95% CI 0·2,15·3) than T- patients. We conclude that islet autoimmunity is much more prevalent in T2D patients than previously reported. T cell-mediated autoimmunity is associated with diminished β-cell function and worse glycemic control.

Identification of a Novel Antigen for Serological Diagnosis of Scrub Typhus

Scrub typhus is an acute infectious disease caused by the bacterium Orientia tsutsugamushi, which is widely distributed in northern, southern, and eastern Asia. Early diagnosis is essential because the average case fatality rate is usually >10% but can be as high as 45% if antimicrobial treatment is delayed. Although an O. tsutsugamushi 56-kDa type-specific antigen (TSA) is commonly used for serological diagnosis of scrub typhus, the 56-kDa TSA shows variations among O. tsutsugamushi strains, which may lead to poor diagnostic results. Therefore, the discovery of new antigenic proteins may improve diagnostic accuracy. In this study, we identified an O. tsutsugamushi 27 kDa antigen through an immunoinformatic approach and verified its diagnostic potential using patient samples. Compared with the O. tsutsugamushi 56-kDa antigen, the new 27-kDa antigen showed better diagnostic specificity with similar diagnostic sensitivity. Therefore, the O. tsutsugamushi 27-kDa antigen shows potential as a novel serological diagnostic antigen for scrub typhus, providing higher diagnostic accuracy for O. tsutsugamushi than the 56-kDa antigen.

First detection of Borrelia and Rickettsia species from Ornithodoros ticks in the Republic of Korea

Ticks are considered important vectors among arthropods and are linked to serious medical and veterinary health problems. In this study, we investigated tick-borne pathogens (TBPs) of Ornithodoros (Carios) sawaii and a newly identified Ornithodoros species from migratory bird nests in the uninhabited islands of the Republic of Korea (ROK). Ticks were collected from seabird nests with soil using a Tullgren funnel. Polymerase chain reaction (PCR) was performed using specific primer sets targeting genes of Borrelia spp., Rickettsia sp., Anaplasma phagocytophilum, Anaplasma bovis, and Bartonella spp. for molecular identification of TBPs, and two pathogens, Borrelia sp. and Rickettsia sp. were detected via PCR. Sequence data were analyzed and a phylogenetic analysis was conducted using the maximum-likelihood method in MEGA v.7. The detection rate of Borrelia sp. in O.(C.) sawaii was 6.8 % (5/74), and that of Rickettsia sp. in O. sawaii and the newly identified Ornithodoros species. was 36.5 % (27/74). Sequencing analysis revealed that the 16S ribosomal (r) RNA and flagellin genes of Borrelia sp., and the citrate synthase (gltA) and 17-kDa antigen gene of Rickettsia sp. were closely phylogenetically related to those of Borrelia turicatae and Rickettsia asembonensis. This is the first report identifying Borrelia sp. and Rickettsia sp. from O. sawaii, and Rickettsia sp. from the newly identified Ornithodoros species in the ROK, and these results imply that soft ticks (O. sawaii, and the newly identified Ornithodoros species) may function as pathogen carriers with important implications for public health throughout their distribution areas in Asia.

Pathogen Carriage by Peri-Domestic Fleas in Western Kenya.

Fleas are carriers for many largely understudied zoonotic, endemic, emerging, and re-emerging infectious disease agents, but little is known about their prevalence and role as a vector in Africa. The aim of this study was to determine the diversity of fleas and the prevalence of infectious agents in them collected from human dwellings in western Kenya. A total of 306 fleas were collected using light traps from 33 human dwellings; 170 (55.56%) were identified as Ctenocephalides spp., 121 (39.54%) as Echidnophaga gallinacea, 13 (4.25%) as Pulex irritans, and 2 (0.65%) as Xenopsylla cheopis. Of the 306 individual fleas tested, 168 (54.9%) tested positive for rickettsial DNA by a genus-specific quantitative real-time PCR (qPCR) assay based on the 17-kDa antigen gene. Species-specific qPCR assays and sequencing revealed presence of Rickettsia asembonensis in 166 (54.2%) and Rickettsia felis in 2 (0.7%) fleas. Borrelia burgdorferi, normally known to be carried by ticks, was detected in four (1.3%) flea DNA preparations. We found no evidence of Yersinia pestis, Bartonella spp., or Orientia spp. Not only were Ctenocephalides spp. the most predominant flea species in the human dwellings, but also almost all of them were harboring R. asembonensis.

Calmodulin is involved in the occurrence of extracellular Ca2+ -dependent full-type hyperactivation in boar ejaculated spermatozoa incubated with cyclic AMP analogs.

In mammals, hyperactivation is essential for sperm fertilization with oocytes in vivo. Two types of hyperactivation "full-type and nonfull-type patterns" can be observed in the spermatozoa from boars, bulls, and mice. We have a hypothesis that the full-type hyperactivation is a physiological (in vivo) pattern and are elucidating its molecular bases. The aims of this study were to detect calmodulin in boar sperm flagella by Western blotting and indirect immunofluorescence and to investigate effects of extracellular Ca2+ and calmodulin antagonists "W-7 and W-5 (W-5; a less potent antagonist)" on the occurrence of full-type hyperactivation in boar spermatozoa. Calmodulin was specifically detected as the 17-kDa antigen in the flagella and postacrosomal region of the heads. Full-type hyperactivation could be induced effectively in the samples incubated with 3.42mM CaCl2 for 120-180min, and it was significantly reduced in the concentration-dependent manners of W-7 and W-5. Suppressing effects of W-7 on the full-type hyperactivation were stronger than those of W-5. These observations indicate that flagellar calmodulin is involved in the occurrence of extracellular Ca2+ -dependent full-type hyperactivation in boar spermatozoa. This is the first indication of the intracellular Ca2+ -sensing molecule which can function in the full-type hyperactivation.

29-kDa: A Potential Candidate for Anti-Tick Vaccine Antigen Source as Immunogenic and Stage Reactive Targeting Hard-Bodied Hyalomma Ticks Ixodidae

The objective of present study is to develop a vaccine against Hyalomma hard-bodied tick and to analyze relevant experimental data on immunized indigenous horse breed Morna. Montanide (ISA-50) adjuvant-based vaccine induced significantly (pandlt;0.02) higher antibody titre through intradermal route with 99.98% vaccine efficacy. Humoral response was determined though indirect ELISA; where peak level of serum antibody was recorded after six weeks interval of post-immunization. Most of hematology and biochemical parameters remained consistent to normal reference values. Our report also indicates a significant percentage decline in the numbers of engorged ticks, eggs mass, eggs number and increased tick rejection. The animal travel history can promote tick burden and is a potential risk factor. Based on biological and hematological parameters, it is possible to conclude that 29-kDa antigen can be used as an effective antigen vaccine candidate to control tick infestation detected through humoral response.

2186. Differentiation of Severe Fever with Thrombocytopenia Syndrome from Scrub Typhus

BackgroundSFTS and scrub typhus have similar clinical features and difficult to differentiate. Thus, a study to develop a scoring system to differentiate between two diseases in a clinical setting before the confirmation of laboratory results was reported by Kim et al. However, the statistical power could be low because of low numbers of cases (21 SFTS, 91 scrub typhus), our study analyzed by increasing the number of cases to overcome these limitations.MethodsWe retrospectively collected data from 183 SFTS and 178 scrub typhus patients who visited the 21 hospitals in South Korea between October, 2013 and November, 2017. The study protocol was approved by the IRB of each institution. SFTS was diagnosed through detection of SFTS viral RNA using RT–PCR. Scrub typhus was diagnosed either detection of 56-kDa antigen of O. tsutsugamushi using nested PCR or ≥ 4 fold rise in IgM or IgG titer using indirect IFA. Statistical analyses were performed by using SPSS and Medcalc.ResultsTo differentiate SFTS from scrub typhus, we applied the scoring system proposed by Kim et al. After multivariable logistic regression, altered mental status, leukopenia, prolonged aPTT, and normal CRP(≤ 1.0 mg/dL) were significantly associated with SFTS compared with scrub typhus. Each variable was scored by 1 point, with a total score of 0–4 points, the optimal cutoff value was > 1 for the ROC curve. A score > 1 had 92% sensitivity, 96% specificity for diagnosis of SFTS, with a ROC AUC of 0.974. Because the sensitivity was less than 95%, we changed the normal CRP criteria to ≤ 3.0 mg/dL. The modified scoring system had 97% sensitivity, 96% specificity for diagnosis of SFTS, with an AUC of 0.983, and it showed a statistically higher accuracy than original scoring system (P = 0.0487). In this study, four factors for predicting SFTS were newly developed: leukopenia, prolonged aPTT, normal CRP (≤3.0 mg/dL), and elevated CK (>1,000 IU/L). Our study scoring system had 97% sensitivity, 98% specificity for diagnosis of SFTS, with an AUC of 0.992, and it showed a statistically higher accuracy than original scoring system (P = 0.0308).ConclusionIn conclusion, we can easily differentiate SFTS from scrub typhus by using our scoring system of leukopenia, prolonged aPTT, normal CRP, and elevated CK in the endemic area. Disclosures All authors: No reported disclosures.

Candidatus Rickettsia senegalensis in Cat Fleas (Siphonaptera: Pulicidae) Collected from Dogs and Cats in Cauca, Colombia.

Rickettsia typhi and Rickettsia felis (Rickettsiales: Rickettsiaceae) are flea-transmitted pathogens. They are important causes of acute febrile illness throughout the world. We, therefore, sought to identify the rickettsial species present in the fleas of dogs and cats in the department of Cauca, Colombia. In this study, we collected 1,242 fleas from 132 dogs and 43 fleas from 11 cats. All fleas were morphologically identified as Ctenocephalides felis (Bouché) adults and organized in pools for DNA extraction (234 pools from dogs and 11 from cats). The gltA gene from rickettsiae was targeted for screening amplification using conventional PCR. In total, 144 of the 245 pools (58.7%) were positive. The positive samples were then processed for the amplification of the 17kDa antigen gene (144/144; 100% positive) and sca5 gene (140/144; 97.2% positive). In addition, restriction enzyme length polymorphism analysis using NlaIV on the amplified product of the sca5 gene demonstrated several organisms: 21/140 (15%) were R. felis, 118/140 (84.3%) were Rickettsia asemboensis, and 1/140 (0.7%) were Candidatus Rickettsia senegalensis. Subsequent sequencing confirmed Candidatus Rickettsia senegalensis in C. felis collected from dogs the first reported from Colombia.

Development of multiplex PCR for neglected infectious diseases.

Scrub typhus, murine typhus, and leptospirosis are widely neglected infectious diseases caused by Orientia tsutsugamushi, Rickettsia typhi, and pathogenic Leptospira spp., respectively. Patients usually present with non-specific symptoms and therefore are commonly diagnosed with acute undifferentiated febrile illness. Consequently, patients face delayed treatment and increased mortality. Antibody-based serological test currently used as gold standard has limitations due to insufficient antibody titers, especially in the early phase of infection. In this study, we aimed to develop multiplex PCR to combine 3 primer pairs that target specific genes encoding 56-kDa TSA of O. tsutsugamushi, 17-kDa antigen of R. typhi, and LipL32 of L. Interrogans and evaluate its performance in comparison to the standard serological tests. Using EDTA blood samples of known patients, the sensitivity and specificity of our multiplex PCR was 100% and 70%, respectively. In addition, the assay was able to diagnose the co-infection of scrub typhus and leptospirosis. The assay may be useful in identifying causative agents during the early phase of these diseases, enabling prompt and appropriate treatment.