Co-Infection of Scrub Typhus and Human Granulocytic Anaplasmosis in Korea, 2006

BACKGROUND: Scrub typhus, severe fever with thrombocytopenia syndrome (SFTS) and human granulocytic anaplasmosis (HGA) are important arthropod-borne infectious diseases in Korea and share a common point that they are transmitted by arthropod bites mostly during outdoor activities and there are considerable overlaps of epidemiologic and clinical features at presentation. We investigated the co-infection of these infections. METHODS: The study subjects were patients with laboratory-confirmed scrub typhus who were enrolled retrospectively in 2006. SFTS virus (SFTSV) infection was confirmed by a reverse transcriptase polymerase chain reaction (PCR) to amplify partial L segment of SFTSV for molecular diagnosis. HGA was confirmed by a nested PCR to amplify 16S rRNA gene of Anaplasma phagocytophilum. Direct sequencing of the positive PCR products was performed. Clinical features of co-infected subjects were described. RESULTS: One-hundred sixty-seven patients with scrub typhus were included in the analysis. Co-infection of A. phagocytophilum was identified in 4.2% of scrub typhus patients (7/167). The route of co-infection was uncertain. The co-infected patients had not different clinical manifestations compared to the patients with scrub typhus only. All the study subjects were negative for SFTSV. CONCLUSION: We found retrospective molecular evidence of the co-infection of scrub typhus and HGA in Korea. HGA may be more prevalent than expected and need to be considered as an important differential diagnosis of febrile patients in Korea.

Molecular Detection and Phylogenetic Analysis of Anaplasma phagocytophilum in Horses in Korea

The identification and characterization of pathogenic and zoonotic tick-borne diseases like granulocytic anaplasmosis are essential for developing effective control programs. The differential diagnosis of pathogenic Anaplasma phagocytophilum and non-pathogenic A. phagocytophilum-like Anaplasma spp. is important for implementing effective treatment from control programs. The objective of the present study was to investigate the prevalence of Anaplasma spp. in horses in Korea by nucleotide sequencing and restriction enzyme fragment length polymorphism assay. Of the 627 horses included in the study, only 1 (0.2%) was infected with A. phagocytophilum. Co-infection with A. phagocytophilumlike Anaplasma spp. was not detected in the study. The 16S rRNA sequence of A. phagocytophilum was similar (99.5–100%) to A. phagocytophilum 16S rRNA isolated from horses in other countries. PCR adapted to amplify A. phagocytophilum groEL and msp2 genes failed to generate amplicons, suggesting genetic diversity in these genes. This study is the first molecular detection of A. phagocytophilum in horses in Korea. Human granulocytic anaplasmosis and animal infection of A. phagocytophilum have been reported in Korea recently. Because of vector tick distribution, global warming, and the increase of the horse industry, horses should be considered as a potential reservoir for A. phagocytophilum, and cross infectivity should be evaluated even though a low prevalence of infection was detected in this study. Furthermore, continuous surveillance and effective control measures for A. phagocytophilum should be established to prevent disease distribution and possible transmission to humans.

Survey and Phylogenetic Analysis of Rodents and Important Rodent-Borne Zoonotic Pathogens in Gedu, Bhutan

Rodents are well-known reservoirs and vectors of many emerging and re-emerging infectious diseases, but little is known about their role in zoonotic disease transmission in Bhutan. In this study, a cross-sectional investigation of zoonotic disease pathogens in rodents was performed in Chukha district, Bhutan, where a high incidence of scrub typhus and cases of acute undifferentiated febrile illness had been reported in people during the preceding 4–6 months. Twelve rodents were trapped alive using wire-mesh traps. Following euthanasia, liver and kidney tissues were removed and tested using PCR for Orientia tsutsugamushi and other bacterial and rickettsial pathogens causing bartonellosis, borreliosis, human monocytic ehrlichiosis, human granulocytic anaplasmosis, leptospirosis, and rickettsiosis. A phylogenetic analysis was performed on all rodent species captured and pathogens detected. Four out of the 12 rodents (33.3%) tested positive by PCR for zoonotic pathogens. Anaplasma phagocytophilum, Bartonella grahamii, and B. queenslandensis were identified for the first time in Bhutan. Leptospira interrogans was also detected for the first time from rodents in Bhutan. The findings demonstrate the presence of these zoonotic pathogens in rodents in Bhutan, which may pose a risk of disease transmission to humans.

Molecular and serological detection of Theileria equi, Babesia caballi and Anaplasma phagocytophilum in horses and ticks in Maranhão, Brazil / Detecção molecular e serológica de Theileria equi, Babesia caballi e Anaplasma phagocytophilum em equinos e carrapatos no Maranhão

Equine piroplasmosis is a tick-borne disease caused by the intraeytrhocytic protozoans Babesia caballi and Theileria equi. It has been reported as a main equine parasitic disease. In addition, Anaplasma phagocytophilum, the causative agent of granulocytic ehrlichiosis, causes a seasonal disease in horses. Both diseases, can be detrimental to animal health. In this sense, blood samples and ticks were collected from 97 horses raised in the microregion of Baixada Maranhense, Maranhão State, Brazil. Serum samples were subjected to Indirect Fluorescence Antibody Test (IFAT) and blood samples and ticks to Polymerase Chain Reaction (PCR) to evaluate the infection by Theileria equi, Babesia caballi and Anaplasma phagocytophilum. The overall seroprevalence was 38.14%, 18.55% and 11.34% for T. equi, B. caballi and A. phagocytophilum, respectively. The results of PCR from blood samples showed 13.40% and 3.09% positive samples to T. equi and B. caballi, respectively. A total of 170 tick specimens were collected and identified as Dermacentor nitens, Amblyomma cajennense sensu lato and Rhipicephalus (Boophilus) microplus. It was detected 2.35% (4/170) and 0.59% (1/170) positive tick samples by PCR for T. equi and B. caballi, respectively. All samples were negative to A. phagocytophilum. No statically difference (p>0.05) was observed when gender, age, use of ectoparasiticide and tick presence were analyzed. A BLASTn analysis of the sequenced samples indicated 97 to 100% similarity with T. equi 18S rRNA gene sequences in GenBank and 98 to 100% with B. caballi. Genetic analysis classified the obtained sequences as T. equi and B. caballi cluster, respectively. It can be concluded that these pathogens occur and are circulating in the studied area.(AU)

A piroplasmose equina é uma doença transmitida por carrapatos causada pelos protozoários intraeritrocitários Babesia caballi e Theileria equi. É relatada como uma doença parasitária comum em equinos. Além disso, Anaplasma phagocytophilum, o agente causal da ehrlichiose granulocítica, causa uma doença sazonal em equinos. Ambas as doenças, podem ser prejudiciais para a saúde animal. Nesse sentido, amostras de sangue e carrapatos foram coletadas de 97 cavalos criados na microrregião da Baixada Maranhense, estado do Maranhão, Brasil. As amostras de soro foram submetidas ao Teste de Imunofluorescência Indireta (RIFI) e amostras de sangue e os carrapatos a Reação da Polimerase em Cadeia (PCR) para avaliar a infecção por Theileria equi, Babesia caballi e Anaplasma phagocytophilum. A prevalência foi de 38,14%, 18,55% e 11,34% para T. equi, B. caballi e A. phagocytophilum, respectivamente. Os resultados da PCR para as amostras de sangue demonstraram 13,40% e 3,09% de positividade para T. equi e B. caballi, respectivamente. Um total de 170 specimens de carrapatos foi coletado e foram identificados Dermacentor nitens, Amblyomma cajennense sensu lato and Rhipicephalus (Boophilus) microplus. Obteve-se 2,35% (4/170) e 0,59% (1/170) positivos por PCR para T. equi e B. caballi, respectivamente. Todas as amostras foram negativas para A. phagocytophilum. Não houve diferença estatística significativa (p>0.05) em relação ao sexo, idade, uso de ectoparasiticida e presença de carrapatos. A análise BLASTn das amostras sequenciadas para gene 18S rRNA indicaram 97 a 100% de similaridade com T. equi e 98-100% com B. caballi no GenBank. Análises genéticas classificaram as sequencias obtidas no mesmo clado que T. equi e B. caballi, respectivamente. Podemos concluir que estes patógenos estão circulando na área de estudo.(AU)

Detection of Anaplasma sp. phylogenetically related to A. phagocytophilum in a free-living bird in Brazil / Detecção de Anaplasma sp. filogeneticamente associado a A. phagocytophilum em uma ave de vida livre no Brasil

Abstract Wild animals play an important role in carrying vectors that may potentially transmit pathogens. Several reports highlighted the participation of wild animals on the Anaplasma phagocytophilum cycle, including as hosts of the agent. The aim of this study was to report the molecular detection of an agent phylogenetically related to A. phagocytophilum isolated from a wild bird in the Midwest of the state of Paraná, Brazil. Fifteen blood samples were collected from eleven different bird species in the Guarapuava region. One sample collected from a Penelope obscura bird was positive in nested PCR targeting the 16S rRNA gene of Anaplasma spp. The phylogenetic tree based on the Maximum Likelihood analysis showed that the sequence obtained was placed in the same clade with A. phagocytophilum isolated from domestic cats in Brazil. The present study reports the first molecular detection of a phylogenetically related A. phagocytophilum bacterium in a bird from Paraná State.

Resumo Animais selvagens possuem participação importante como carreadores dos vetores responsáveis por transmitir doenças e vários relatos destacam a participação de animais silvestres no ciclo do Anaplasma phagocytophilum, inclusive como hospedeiros do agente. O presente trabalho tem por objetivo relatar pela primeira vez a detecção molecular da infecção por um agente filogeneticamente associado a A. phagocytophilum em uma ave silvestre no interior do Paraná, Brasil. Foram colhidas 15 amostras de sangue originadas de onze espécies diferentes de aves, todas provenientes da região de Guarapuava. Apenas uma amostra pertencente a uma ave da espécie Penelope obscura foi positiva para o ensaio de nested PCR baseado no gene 16S rRNA. A árvore filogenética baseada na análise por máxima verossimilhança demonstrou que a sequência obtida no presente estudo se posicionou no mesmo clado com cepas de A. phagocytophilum isoladas de gatos domésticos no Brasil. O presente trabalho relata pela primeira vez a detecção molecular de Anaplasma sp. filogeneticamente relacionado à A. phagocytophilum, em um animal da espécie P. obscura, assim como a presença do parasita em uma ave silvestre do Estado do Paraná, Brasil.

Important frequency of Anaplasma phagocytophilum infection in a population of domiciled dogs in an urbanized area in south-eastern Brazil / Importante frequência da infecção por Anaplasma phagocytophilum em uma população de cães domiciliados em área urbanizada no sudeste do Brasil

Anaplasma phagocytophilum is responsible for granulocytic anaplasmosis in humans and various animal species. The aim of the present study was to determine the prevalence of A. phagocytophilum-infected dogs in a residential area of Belo Horizonte, Minas Gerais state, Brazil. A total of 62 dogs were submitted to serological (indirect fluorescent-antibody -IFI) and molecular (PCR) tests. Anti-A. phagocytophilum antibodies were detected in 43.8% of the dogs. Seven dogs (10.9%) were PCR-positive for the msp4 gene, six and four of these were positive for the for the msp2/p44 gene of A. phagocytophilum and 16S rRNA region of granulocytic Anaplasmataceae respectively. This study confirms a relatively high frequency of A. phagocytophilum infection in a population of domiciled dogs in an urbanized area in south-eastern Brazil and highlights the need for further studies on the role of Rhipicephalus sanguineus sensu lato ticks in the transmission of this bacterium to dogs in urban Brazilian areas.(AU)

Anaplasma phagocytophilum é responsável pela anaplasmose granulocítica, doença que acomete seres-humanos e várias espécies de animais. O objetivo do presente estudo foi determinar a prevalência de cães acometidos por A. phagocytophlium em uma área residencial de Belo Horizonte, MG, Brasil. Sessenta e dois cães foram submetidos a testes sorológicos (reação de imunofluorescência indireta - IFAT) e moleculares (PCR). Anticorpos anti-A. phagocytophilum foram detectados em 43,8% dos cães. Sete cães (10,9%) foram positivos no PCR para o gene msp4 de A. phagocytophilum, seis para o gene msp2/p44 A. phagocytophilum e quatro para a região 16S rRNA de Anaplasmataceae granulocíticas. Esse estudo confirma a frequência relativamente alta da infecção por A. phagocytophilum em uma população de cães domiciliados em área urbanizada no sudeste do Brasil e destaca a necessidade de pesquisas para determinar o papel do carrapato Rhipicephalus sanguineus sensu lato na transmissão desse microrganismo para cães de áreas urbanas brasileiras.(AU)

Prevalence of Anaplasma, Bartonella and Borrelia Species in Haemaphysalis longicornis collected from goats in North Korea

North Korea is located on the northern part of the Korean Peninsula in East Asia. While tick-borne pathogens of medical and veterinary importance have been reported from China and South Korea, they have not been reported from North Korea. To screen for zoonotic tick-borne pathogens in North Korea, ticks were collected from domestic goats. A total of 292 (27 nymph, 26 male, 239 female) Haemaphysalis (H.) longicornis were collected and assayed individually for selected tick-borne pathogens. A total of 77 (26.4%) were positive for Anaplasma bovis, followed by Bartonella (B.) grahamii (15, 5.1%), Anaplasma phagocytophilum (12, 4.1%), Bartonella henselae (10, 3.4%), and Borrelia spp. (3, 1.0%) based on 16S ribosomal RNA and ITS species-specific nested polymerase chain reaction. Using the groEL-based nested PCR, a total of 6 and 1 H. longicornis were positive for B. grahamii and B. henselae, respectively. All products were sequenced and demonstrated 100% identity and homology with previously reported sequences from other countries in GenBank. This is the first report of the detection of tick-borne pathogens in the North Korea and suggests that farm animals may act as reservoirs for zoonotic tick-borne pathogens.

Tick-borne rickettsial pathogens in questing ticks, removed from humans and animals in Mexico

Tick-borne rickettsial diseases (TBRD) are commonly encountered in medical and veterinary clinical settings. The control of these diseases is difficult, requiring disruption of a complex transmission chain involving a vertebrate host and ticks. The geographical distribution of the diseases is related to distribution of the vector, which is an indicator of risk for the population. A total of 1,107 ticks were collected by tick dragging from forests, ecotourism parks and hosts at 101 sites in 22 of the 32 states of Mexico. Collected ticks were placed in 1.5 mL cryovials containing 70% ethanol and were identified to species. Ticks were pooled according to location/host of collection, date of collection, sex, and stage of development. A total of 51 ticks were assayed by polymerase chain reaction (PCR) to confirm species identification using morphological methods. A total of 477 pools of ticks were assayed using PCR techniques for selected tick-borne pathogens. Anaplasma phagocytophilum was the most commonly detected pathogen (45 pools), followed by, Ehrlichia (E.) canis (42), Rickettsia (R.) rickettsii (11), E. chaffeensis (8), and R. amblyommii (1). Rhipicephalus sanguineus was the tick most frequently positive for selected pathogens. Overall, our results indicate that potential tick vectors positive for rickettsial pathogens are distributed throughout the area surveyed in Mexico.

Molecular and phylogenetic analysis of Anaplasma spp. in sheep and goats from six provinces of China

Members of the genus Anaplasma are important emerging tick-borne pathogens in both humans and animals in tropical and subtropical areas. Here, we investigated the presence of Anaplasma spp. in 621 sheep and 710 goats from six provinces of China. Polymerase chain reaction (PCR) and DNA sequencing were conducted to determine the prevalence of Anaplasma (A.) phagocytophilum, A. ovis and A. bovis targeting the 16S ribosomal RNA or the major surface protein 4 gene. PCR revealed Anaplasma in 39.0% (240/621) of sheep and 45.5% (323/710) of goats. The most frequently detected species was A. ovis (88/621, 14.2% for sheep; 129/710, 18.2% for goats), followed by A. bovis (60/621, 9.7% for sheep; 74/710, 10.4% for goats) and A. phagocytophilum (33/621, 5.3% for sheep; 15/710, 2.1% for goats). Additionally, eight sheep and 20 goats were found to be infected with three pathogens simultaneously. DNA sequencing confirmed the presence of these three Anaplasma species in the investigated areas, and phylogenetic analysis indicated that there was geographic segregation to a certain extent, as well as a relationship between the host and cluster of A. ovis. The results of the present study provide valuable data that helps understand the epidemiology of anaplasmosis in ruminants from China.

Thrombocyte indices in dogs infected with Ehrlichia canis and Anaplasma phagocytophilum / Índices de trombocitos en perros infectados con Ehrlichia canis y Anaplasma phagocytophilum

Objective. In the present study alterations in trombocyte numbers and trombocyte indices were investigated in 51 dogs naturally infected with E. canis and/or A. phagocytophilum. Achieved results were compared to those of 20 healty dogs comprising control group. Materials and methods. Naturally occuring vector borne diseases were diagnosed by use of a canine point-of-care ELISA kit (Snap 4Dx, Idexx). Dogs were enrolled into 3 groups as follows; II. group involved A. phagocytophilum infected dogs (n=10), III. group (n=13) E. canis+ A. phagocytophilum co-infected, and IV. group (n=28) E. canis infected dogs. Healthy controls (n=20) were enrolled in group I. Results. Mean PLT counts were significantly decreased in II., III. and IV. groups (159.6±63.5, 142.3±44.3, 148.7±33.5, respectively) in comparison to control group (370.4±28.6) (p≤0.01). Mean PCT values in groups II., III. and IV. (0.1530±0.590, 0.1531±0.0441, 0.1450±0.314, respectively) were significantly decreased in contrast to control group (0.3695±0.0283) (p≤0.01). Between PLT and PCT values, statistically significant positive correlation (p≤0.01) (r=0.988, 0.990 and 0.981, respectively) was evident among groups II., III. and IV. Conclusions. Infected dogs showed significant alterations (p≤0.01) among mean PLT and PCT values and a positive correlation was evident between those 2 parameters (p≤0.01), whereas alterations on mean MPV and PDWc were not statistically significant. Finally it was suggested that according to the aforimentioned results, PLT and PCT values may be used as valuable parameters for diagnosis and probably for monitorization and prognosis in infected dogs with Ehrlichiosis and/or Anaplasmosis.

Objetivo. Comparar las alteraciones en el número de trombocitos y en los índices de trombocitos en perros infectados naturalmente con E. canis y A. phagocytophilum. Materiales y métodos. Los perros fueron distribuidos en cuatro grupos: Grupo I; perros sanos (n=20), Grupo II; perros infectados con A. phagocytophilum (n=10), Grupo III; perros infectados con E. canis + A. phagocytophilum (n=13) y grupo IV; perros infectados con E. Canis (n=28). Resultados. Los recuentos de PLT se redujeron significativamente (p≤0.01) en los grupos II, III y IV (159.6±63.5, 142.3±44.3, 148.7±33.5, respectivamente) en comparación con el grupo control (370.4±28.6). La media de los valores de PCT en los grupos II, III y IV fue de 0.1530±0.590, 0.1531±0.0441 y 0.1450±0.314 respectivamente, cuyas reducciones fueron significativas (p≤0.01) en contraste con el grupo control (0.3695±0.0283). Entre los valores de PLT y el PCT la correlación fue positiva (r =0.988) y significativa (p≤0.01), 0.990 y 0.981, respectivamente, entre los grupos II, III y IV. Conclusiones. Los perros infectados mostraron alteraciones significativas (p≤0.01) entre PLT y los valores del PCT, así como una correlación positiva entre esos dos parámetros (p≤0.01), mientras que las alteraciones de MPV y PDWc no fueron significativas. Se sugiere que de acuerdo con los resultados obtenidos, los valores de PLT y el PCT se pueden utilizar como parámetros valiosos para el diagnóstico y probablemente, para el monitoreo y el pronóstico en perros infectados con la Ehrlichiosis o la Anaplasmosis.

Spatial distribution of vector borne disease agents in dogs in Aegean region, Turkey / Distribución espacial de agentes patógenos transmitidos por vectores en perros en la región del Egeo, Turquía

Objective. Assess the spatial distribution of seroprevalence of infection with or exposure to 4 vector-borne pathogens Ehrlichia canis, Anaplasma phagocytophilum, Borrelia burgdorferi and Dirofilaria immitis, across the coastal states of the Aegean region with special reference to clinical signs and haematological variances related to disease condition. Materials and methods. A convenience sample, targeting blood from at least 10 pet dogs from Izmir, Aydin, Denizli, Mugla and Manisa cities involved was evaluated using a canine point-of-care ELISA kit. Results. Out of 307 dogs tested the overall seroprevalence was highest for E. canis (24.42%), followed by E. canis + A. phagocytophilum co-infection (10.42%), A. phagocytophilum (7.49%) and D. immitis (2.28%). Only 2 cases were seropositive to B. burgdorferi albeit 10 dogs were co-infected with more than 2 agents. For both dogs infected with E. canis and co-infected with E. canis and A. phagocytophilum, anemia, thrombocytopenia and leukocytosis, were more commonly detected, whereas thrombocytopenia and leukocytosis were significant finding in dogs infected with A. phagocytophilum or D. immitis, respectively. Variance analysis showed significant differences for mean RBC, Hb, PCV and PLT values (p<0.01) among control group and other groups. Conclusions. Seropositivity for vector-borne pathogens other than B. burgdorferi, is moderately to widely distributed in dogs residing in the Aegean region in Turkey.

Objetivo. Evaluar la distribución espacial de la seroprevalencia de la infección de 4 agentes patógenos de transmisión por vectores Ehrlichia canis, Anaplasma phagocytophilum, Borrelia burgdorferi y Dirofilaria immitis, en los estados costeros de la región del Egeo con especial referencia a los signos clínicos y las variaciones hematológicas relacionadas con la enfermedad. Materiales y métodos. Se tomaron por conveniencia muestras de sangre de al menos 10 perros en las ciudades Izmir, Aydin, Denizli, Mugla y Manisa. Para la evaluación de las muestras se utilizó un kit de ELISA para la detencción de anticuerpos de las enfermedades del estudio. Resultados. De los 307 perros muestreados, la seroprevalencia más alta fue para E. canis (24.42%), seguido por la coinfección entre E. canis + A. phagocytophilum (10.42%), A. phagocytophilum (7.49%) y D. immitis (2.28%). Sólo 2 casos fueron seropositivos para B. burgdorferi aunque 10 perros fueron coinfectados con más de 2 agentes. En ambos perros infectados con E. canis y coinfectados con E. canis y A. phagocytophilum, se detectó comúnmente anemia, trombocitopenia y leucocitosis, mientras que la trombocitopenia y leucocitosis fueron significativos en perros infectados con A. phagocytophilum o D. immitis , respectivamente. El análisis de varianza mostró diferencias significativas para los promedios de RBC, hemoglobina, hematocrito y valores PLT (p<0.01) entre el grupo control y los otros grupos. Conclusiones. La seropositividad transmitida por vectores patógenos distintos de B. burgdorferi, fue moderada y ampliamente distribuida en los perros que residen en la región del Egeo en Turquía.

Multispacer typing of Chinese Anaplasma phagocytophilum isolates / 中华流行病学杂志

<p><b>OBJECTIVE</b>To type the Chinese Anaplasma phagocytophilum isolates by Multispacer typing (MST).</p><p><b>METHODS</b>Based on the genomes of the 4 published Anaplasma strains, 4 genomic sequences were analyzed by Mauve 2.3.1 software and variable spacer sequences were selected for designing primers with the bio-software Primer Premier 5.0. A total of 11 Chinese A. phagocytophilum isolates, obtained from different areas of China during 2009-2012 were assayed by the MST. Twenty two intergenic sequences for each isolate tested and the reference A. phagocytophilum strain Webster and A. phagocytophilum strain HZ were concatenated in the order of HGA-mst 1F/1R-mst 2F/2R, HGA-mst 22F/22R.</p><p><b>RESULTS</b>Twenty two pairs of primers were successfully used for typing the Human granulocytic anaplasmosis (HGA) strains in the study. Those 22 intergenic sequences exhibited a great diversity among the strains tested and each of the strain tested was identified as unique genotype, according to the alignment analysis of the 22 concatenated intergenic sequences. Of these single nucleotide polymorphism (SNPs) identified in the study, the nucleotide transitions shared the highest percentage (60.2%, 251/417) and then the nucleotide transversion, accounted for 23.0% (96/417) and the indel events (insertion/deletion) were observed of 16.7% (70/417)SNPs. Phylogenetic analysis indicated that the 5 strains from patients (LZ-H1, LZ-H2, LZ-H3, LZ-H4, LZ-H5) from Laizhou areas, Shandong province and 1 tick strain (LZ-T1) from Haemaphysalis longicornis collected from the same areas where the patients lived were grouped in the same clan with the reference A. phagocytophilum strain Webster and strain HZ. Beijing isolates (BJ-H1) grouped with Xinjiang isolates (XJ-H1 and XJ-H3) while another tick isolates from Laizhou areas (LZ-T2) and another Xinjiang human isolate(XJ-H2)were in the same clan, which was closely related to the isolates from severe patients in Laizhou.</p><p><b>CONCLUSION</b>Chinese HGA isolates exhibited a great diversity of intergenic regions. MST seemed a valuable tool for the detection and tracing for any endemic strains of Anaplasma during the outbreak investigations in the public health events.</p>

Seroepidemiological investigation of lyme disease and human granulocytic anaplasmosis among people living in forest areas of eight provinces in China / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>Lyme disease and Human granulocytic anaplasmosis are tick-borne diseases caused by Borrelia burgdorferi and Anaplasma phagocytophilum respectively. We have investigated infection and co-infection of the two diseases in the population of forest areas of eight provinces in China by measuring seroprevalence of antibodies against B. burgdorferi and A. phagocytophilum.</p><p><b>METHODS</b>Forest areas in 8 provinces were chosen for investigation using whole sampling and questionnaire survey methods. 3 669 serum samples from people in the forest areas were tested for the presence of antibodies by indirect immunofluorescent assay (IFA).</p><p><b>RESULTS</b>Seroprevalence against B. burgdorferi was 3% to 15% and against A. phagocytophilum was 2% to 18% in the study sites in the 8 provinces in China. We also found co-infection of B. burgdorferi and A. phagocytophilum in 7 of the 8 provinces (the exception being the Miyun area in Beijing). The seroprevalence for both B. burgdorferi and A. phagocytophilum was significantly higher among people exposed to ticks than among people who were not exposed to ticks.</p><p><b>CONCLUSION</b>We conclude that both pathogens are endemic in the forest areas in the eight provinces, but the prevalence of B. burgdorferi and A. phagocytophilum differs between the provinces.</p>

In vitro cultivation of Anaplasma marginale and A. phagocytophilum in tick cell lines: a review / Cultivo in vitro de Anaplasma marginale e A. phagocytophilum em células de carrapatos: uma revisão

Continuous cell lines have been established from several ixodid and argasid tick species, representing an excellent tool suitable for the isolation of pathogens and their subsequent propagation, which in turn allows the production of antigenic material for diagnostic tests, antibody and vaccine production, and also for studies on host-vector-pathogen relationships. This paper reviews the use of tick cells for culture initiation and maintenance of two obligate intracellular bacterial pathogens, Anaplasma marginale and Anaplasma phagocytophilum. These in vitro cultivation systems have been used in a wide range of studies, covering morphological ultrastructural analysis, genetics, proteomics and biological differences between strains, including genome transcriptional and protein expression approaches, enabling comparisons between host and vector cells. Thus, such systems open a new window for a better understanding of interactions between pathogens and tick cells. Last but not least, such systems contribute to the reduction in usage of animals for experimental research, as antigenic material can be produced in reasonably large quantities without the use of in vivo species-specific systems.

Linhagens contínuas de células já foram estabelecidas a partir de várias espécies de carrapatos ixodídeos e argasídeos e representam uma ferramenta excelente para o isolamento e propagação de patógenos, permitindo a produção de material antigênico para testes diagnósticos, produção de anticorpos e vacinas, e também para estudos das relações entre hospedeiro-vetor-patógenos. Este artigo revisa o uso de células de carrapatos para estabelecimento e manutenção in vitro de dois patógenos intracelulares, Anaplasma marginale e Anaplasma phagocytophilum. Estes sistemas de cultivo in vitro, têm sido utilizados em vários estudos, tais como análises morfológicas, genéticas, proteômicas e estudos diferenciais entre isolados, incluindo genômica transcricional e expressões proteicas, permitindo comparações entre células dos hospedeiros e dos vetores. Tais sistemas constituem, portanto, uma nova abordagem para melhor entendimento das relações entre patógenos e células de carrapatos. Além disso, tais sistemas contribuem para a redução do uso de animais de experimentação, uma vez que permitem a produção de grandes quantidades de material antigênico sem o uso de sistemas espécie-específicos in vivo.

Serological investigation of vector-borne disease in dogs from rural areas of China

<p><b>OBJECTIVE</b>To evaluate the Anaplasma phagocytophilum (A. phagocytophilum), Ehrlichia canis (E. canis), Dirofilaria immitis (D. immitis) (canine heartworm), Borrelia burgdorferi (B. burgdorferi) infections in countryside dogs from Yunnan, Hainan and Anhui provinces.</p><p><b>METHODS</b>Serum samples were collected from 26 dogs in Yunnan, Hainan and Anhui provinces. The samples were tested using a commercial ELISA rapid diagnostic assay kit (SNAP(®) 4Dx(®); IDEXX Laboratories, Inc. U.S.A.). Meanwhile, indirect immunofluorescence assay (IFA) recommended by WHO was conducted to detect IgG to A. phagocytophilum. Two methods were analyzed and compared.</p><p><b>RESULTS</b>The number of serologically positive dogs for IgG to A. phagocytophilum was only 2 which was from Hainan province and none of the 26 dogs responded positive for E. canis, D. immitis (canine heartworm), and B. burgdorferi by ELISA rapid diagnostic method. The number of serologically positive dogs for IgG to A. phagocytophilum was 13 (50%) by IFA method. Data of the two methods were analyzed by statistical software and the difference was statistically significant (P=0.002).</p><p><b>CONCLUSIONS</b>It can be concluded that IFA method was more sensitive than ELISA rapid diagnostic method. However, we need conduct further and intensive epidemiology survey on tick-born diseases pathogens including A. phagocytophilum, E. canis, D. immitis (canine heartworm), and B. burgdorferi which have public health significance.</p>

Identification of Anaplasma phagocytophilum in small mammals from Hengduan Mountains of Southwest China / 中华流行病学杂志

<p><b>OBJECTIVE</b>To investigate the prevalence of Anaplasma phagocytophilum in small mammals from the forest area of Hengduan Mountains in southwestern China.</p><p><b>METHODS</b>Small mammals captured from Gaoligong and Xianggelila mountainous area of Yunnan province were detected by PCR amplification. The sequences of 16S rRNA and Msp4 gene fragments from positive samples were compared with corresponding sequences deposited in GenBank.</p><p><b>RESULTS</b>A total number of 436 small animals, which belongs to 5 orders 18 genera 35 species were tested, 32 (7.34%) were positive in 6 genera 11 species. There were 8.64% (26/301) positive in 25 species at Goligong mountainous areas, and 4.44% (6/135) were positive in 19 species at the Xianggelila mountainous areas. Positive small mammals were most rodents. The nucleotide sequences of A.phagocytophilum 16S rRNA gene amplified from small mammals varied from 99% - 100% and were 99% - 100% similar with the corresponding segments of A. phagocytophilum from Jilin deposited in GeneBank. The sequences of A. phagocytophilum Msp4 gene showed that there was 95% - 97% similarity with the corresponding sequences registered in GenBank.</p><p><b>CONCLUSION</b>A. phagocytophilum was firstly identified in 6 genera 11 species small mammals from a forest area of Hengduan Mountainous areas in southwestern China. Rodents might serve as the primary hosts indicating the potential risk to the domestic animals and human beings in this area.</p>

Genetic Identification and Phylogenetic Analysis of Anaplasma and Ehrlichia Species in Haemaphysalis longicornis Collected from Jeju Island, Korea

A total of 1,395 Haemaphysalis longicornis ticks collected from Jeju Island of Korea were examined by 16S rRNA gene-based nested PCR for the presence of infection with Anaplasma and Ehrlichia species. Template DNAs to detect the tick-borne pathogens were prepared from a total 506 tick pools. Eight genera of Anaplasma and six Ehrlichia by 16S rRNA gene PCR and sequencing analysis were identified. A. phagocytophilum was the most prevalent (27 [1.9%]) by nested PCR, followed by A. bovis (5 [0.4%]), E. chaffeensis (4 [0.2%]), and A. centrale (1 [0.1%]). In the phylogenetic analysis based on 16S rRNA sequences, eight genera of Anaplasma group (> 99.4% homology) and six Ehrlichia group (> 99.5% homology) were close to deposited A. marginale strains (AF309867, AF414874, and FJ226454) and Ehrlichia sp. (DQ324547), respectively. Three Anaplasma species groups A. phagocytophilum (group A), A. bovis (group B), and A. centrale (group C) and one Ehrlichia species E. chaffeensis (group D) were determined by comparing with Anaplasma and Ehrlichia related sequences. First, twenty-eight A. phagocytophilum clones belonging to group A were divided into 7 genotypes. The sequence similarity among genotypes A1 to A4 was very high (> 99.6%). Genotype B2 was close to A. bovis from Korea (99.7%). Genotype D1 was close to known E. chaffeensis strains (M73222, AF147752, and AY350424) and their similarity value was 99.7%. In conclusion, the genera of Anaplasma/Ehrlichia, A. phagocytophilum, and E. chaffeensis identified in predominant H. longicornis ticks were ubiquitous throughout the Jeju Island. The various native groups have been found through sequence identities and phylogenetic analysis.

Microbial pathogens in ticks, rodents and a shrew in northern Gyeonggi-do near the DMZ, Korea

A total of 1,618 ticks [420 individual (adults) and pooled (larvae and nymphs) samples], 369 rodents (Apodemus arius, Rattus norvegicus, Tscherskia triton, Mus musculus, and Myodes regulus), and 34 shrews (Crocidura lasiura) that were collected in northern Gyeonggi-do near the Demilitarized Zone (DMZ) of Korea during 2004-2005, were assayed by PCR for selected zoonotic pathogens. From a total of 420 individual and pooled tick DNA samples, Anaplasma (A.) phagocytophilum (16), A. platys (16), Ehrlichia (E.) chaffeensis (63), Borrelia burgdorferi (16), and Rickettsia spp. (198) were detected using species-specific PCR assays. Out of 403 spleens from rodents and shrews, A. phagocytophilum (20), A. platys (34), E. chaffeensis (127), and Bartonella spp. (24) were detected with species-specific PCR assays. These results suggest that fevers of unknown causes in humans and animals in Korea should be evaluated for infections by these vector-borne microbial pathogens.

Evidencia clínica y serológica de rickettsiosis canina en Chile / Clinical and serological evidence of canine rickettsiosis in Chile

En Chile, no se han documentado infecciones por Rickettsias en mascotas; algunas de ellas tienen importante potencial zoonótico. Objetivos: Reportar dos casos de rickettsiosis canina con confirmación serológica y determinar seroprevalencia de Rickettsia sp un grupo de caninos. Métodos: IgG anti-Rickettsia conorii y anti-Anaplasma phagocitophilum por IFI en dos caninos con cuadro clínico sugerente de rickettsiosis. Determinación de IgG anti-R. conorii en 77 caninos. Resultados: Como casos clínicos hubo un canino con fiebre, mialgias y melena y otro con manifestaciones he-morrágicas y compromiso neurológico. Seroprevalencia: 35% de los caninos presentaban IgG anti-Rickettsia. Discusión: Se reporta por primera vez en Chile la presencia de rickettsiosis canina, tanto clínica como serológica. Se documenta co-infección por Rickettsia y Anaplasma, dos agentes transmitidos por garrapatas. Es necesario realizar estudios de biología molecular para confirmar la especie de rickettsia presente en Chile. Además, debe estudiarse el rol zoonótico de estas infecciones en nuestro medio.

Rickettsial infections in pets have not been documented in Chile. Some of those infections have relevant zoonotic potential. Objectives: To report two serologically confirmed cases of canine rickettsiosis. To determine seroprevalence to Rickettsia sp in a group of dogs. Methods: IgG antibodies anti-R. conorii and anti-A. phagocitophilum by IFI in two dogs with clinical rickettsiosis. IgG antibodies anti-R. conorii in a group of 77 dogs. Results: Clinical cases: a dog presented with fever, myalgias and melena, another dog with bleeding and neurological involvement. Seroprevalence: 35% of the dogs had antibodies against Rickettsia. Discussion: This is the first evidence of canine rickettsiosis in Chile, both clinical and serological. Co-infection with two tickborne agents: Rickettsia and Anaplasma, is documented. Molecular studies are needed to confirm the rickettsial species present in Chile. The zoonotic role of these infections must be also studied.