A total of 1,708 small mammals (1,617 rodents and 91 soricomorphs), including Apodemus agrarius (n = 1,400), Microtus fortis (167), Crocidura lasiura (91), Mus musculus (32), Myodes (= Eothenomys) regulus (9), Micromys minutus (6), and Tscherskia (= Cricetulus) triton (3), were live-trapped at US/Republic of Korea (ROK) military training sites near the demilitarized zone (DMZ) of Paju, Pocheon, and Yeoncheon, Gyeonggi Province from December 2004 to December 2009. Small mammals were examined for their intestinal nematodes by necropsy. A total of 1,617 rodents (100%) and 91 (100%) soricomorphs were infected with at least 1 nematode species, including Nippostrongylus brasiliensis, Heligmosomoides polygyrus, Syphacia obvelata, Heterakis spumosa, Protospirura muris, Capillaria spp., Trichuris muris, Rictularia affinis, and an unidentified species. N. brasiliensis was the most common species infecting small mammals (1,060; 62.1%) followed by H. polygyrus (617; 36.1%), S. obvelata (370; 21.7%), H. spumosa (314; 18.4%), P. muris (123; 7.2%), and Capillaria spp. (59; 3.5%). Low infection rates (0.1-0.8%) were observed for T. muris, R. affinis, and an unidentified species. The number of recovered worms was highest for N. brasiliensis (21,623 worms; mean 20.4 worms/infected specimen) followed by S. obvelata (9,235; 25.0 worms), H. polygyrus (4,122; 6.7 worms), and H. spumosa (1,160; 3.7 worms). A. agrarius demonstrated the highest prevalence for N. brasiliensis (70.9%), followed by M. minutus (50.0%), T. triton (33.3%), M. fortis (28.1%), M. musculus (15.6%), C. lasiura (13.2%), and M. regulus (0%). This is the first report of nematode infections in small mammals captured near the DMZ in ROK.
Nippostrongylus brasiliensis; Heligmosomoides polygyrus; Syphacia obvelata; Heterakis spumosa; Protospirura muris; Capillaria spp.; Trichuris muris; Rictularia affinis; nematode; rodent; insectivore
In order to investigate the prevalence of tick-borne infectious agents among ticks, ticks comprising five species from two genera (Hemaphysalis spp. and Ixodes spp.) were screened using molecular techniques. Ticks (3,135) were collected from small wild-caught mammals or by dragging/flagging in the Republic of Korea (ROK) and were pooled into a total of 1,638 samples (1 to 27 ticks per pool). From the 1,638 tick samples, species-specific fragments of Anaplasma phagocytophilum (1 sample), Anaplasma platys (52 samples), Ehrlichia chaffeensis (29 samples), Ehrlichia ewingii (2 samples), Ehrlichia canis (18 samples), and Rickettsia rickettsii (28 samples) were amplified by PCR assay. Twenty-one pooled and individual tick samples had mixed infections of two (15 samples) or three (6 samples) pathogens. In addition, 424 spleen samples from small captured mammals (389 rodents, 33 insectivores, and 2 weasels) were screened for selected zoonotic pathogens. Species-specific DNA fragments of A. phagocytophilum (110 samples), A. platys (68 samples), E. chaffeensis (8 samples), E. ewingii (26 samples), E. canis (51 samples), and Rickettsia sp. (22 samples) were amplified by PCR assay. One hundred thirty small mammals had single infections, while 4, 14, and 21 striped field mice (Apodemus agrarius) had mixed infections of four, three, and two pathogens, respectively. Phylogenetic analysis based on nucleotide sequence comparison also revealed that Korean strains of E. chaffeensis clustered closely with those from China and the United States, while the Rickettsia (rOmpA) sequences clustered within a clade together with a Chinese strain. These results suggest that these agents should be considered in differential diagnosis while examining cases of acute febrile illnesses in humans as well as animals in the ROK.
Comprehensive quarterly serosurveillance on scrub typhus in small mammals collected from military training sites located near the Demilitarized Zone (DMZ), northern Gyeonggi-do (Province), ROK was conducted to determine the potential rodent-borne and associated ectoparasite disease risks to military personnel. A total of 1,196 rodents and insectivores representing 8 species, Apodemus agrarius (87.3%, n = 1,044), Mus musculus (5.4%, n = 65), Crocidura lasiura (3.3%, n = 40), Microtus fortis (2.6%, n = 31), Micromys minutus (0.3%, n = 4), Tscherskia triton (0.3%, n = 4), Rattus norvegicus (0.3%, n = 4), and Myodes regulus (0.3%, n = 4) were assayed for the presence of antibodies to Orientia tsutsugamushi. O. tsutsugamushi antibodies were detected in 6 of 8 species and seroprevalence determined; A. agrarius (45.6%), M. musculus (23.1%), M. fortis (48.4%), M. minutus (50.0%), T. triton (50.0%), and R. norvegicus (25.0%). A total of 31,184 chigger mites collected from 508 rodents and insectivores were slide-mounted and 10 species belonging to 4 genera were identified. Leptotrombidium pallidum (53.4%) was the most frequently collected, followed by L. palpale (15.7%), Neotrombicula tamiyai (14.3%), L. orientale (10.7%), L. zetum (3.1%), Walchia fragilis (2.1%), and L. gemiticulum (0.8%), while the remaining 3 species, L. subintermedium, N. gardellai, and Euschoengastia koreaensis were rarely observed (prevalence < 10%). In contrast to previous surveys, higher chigger indices of the primary scrub typhus vectors, L. pallidum (165.4), L. orientale (45.0), and L. palpale (21.4), were observed during the spring season.
Apodemus agrarius; Mus musculus; Crocidura lasiura; chigger; Leptotrombidium; scrub typhus
A total of 1,498 small mammals (rodents and insectivores), including Apodemus agrarius (n = 1,366), Crocidura lasiura (54), Mus musculus (32), Micronytus fortis (28), Eothenomys regulus (9), Micronys minutes (6), and Cricetulus triton (3), were live-trapped in Gyeonggi-do (Province) (Paju-si, Pocheon-gun, and Yeoncheon-gun) near the demilitarized zone (DMZ) from December 2004 to September 2005. A. agrarius was found to be infected with 3 species of echinostomes (Echinostoma hortense, Echinostoma cinetorchis, and Euparyphium murinum), while C. lasiura was infected with 1 species (Echinochasmus japonicas) of echinostome. Other mammals were free from echinostome infections. Total 16 E. hortense were detected in 7 (0.5%) mice, 9 E. cinetorchis from 5 (0.4%), and 3 E. murinum from 2 (0.1%) out of 1.366 A. agrarius examined. E. japonicus was found only in 1 (1.9%; total 3 specimens) C. lasiura. These results demonstrate that A. agrarius and C. lasiura, inhabiting near the DMZ of Gyeonggi-do serve as the natural definitive hosts for several species of echinostomes, although their infection rates are low. This is the first record of natural infections of A. agrarius with E. cinetorchis and C. lasiura with E. japonicus in the Republic of Korea.
Echinostoma hortense; Echinostoma cinetorchis; Euparyphium murinum; Echinochasmus japonicus; striped field mouse; Apodemus agrarius; shrew; Crocidura lasiura; DMZ
Rodent-borne disease surveillance was conducted at Nightmare Range (NM-R), near the demilitarized zone in northeast Gyeonggi Province, Republic of Korea, to identify hemorrhagic fever with renal syndrome (HFRS) risks for a mountainous high-elevation (500 m) military training site. Monthly surveys were conducted from January 2008-December 2009. A total of 1,720 small mammals were captured belonging to the Orders Rodentia [Families, Sciuridae (1 species) and Muridae (7 species)] and Soricomorpha [Family, Soricidae (1species)]. Apodemus agrarius, the primary reservoir for Hantaan virus (HTNV), accounted for 89.9% (1,546) of all small mammals captured, followed by Myodes regulus (4.0%), Crocidura lasiura (3.9%), Micromys minutus (1.4%), Mus musculus (0.3%), Microtus fortis (0.2%), Apodemus peninsulae (0.2%), Tamias sibiricus (0.1%), and Rattus norvegicus (<0.1%). Three species were antibody-positive (Ab+) for hantaviruses: A. agrarius (8.2%), M. minutus (4.2%), and C. lasiura (1.5%). HTNV specific RNA was detected in 93/127 Ab+ A. agrarius, while Imjin virus specific RNA was detected in 1/1 Ab+ C. lasiura. Overall, hantavirus Ab+ rates for A. agrarius increased with weight (age) and were significantly higher among males (10.9%) than females (5.1%) (P<0.0001). High A. agrarius gravid rates during the fall (August-September) were associated with peak numbers of HFRS cases in Korea that followed high gravid rates. From 79 RT-PCR positive A. agrarius, 12 HTNV RNA samples were sequenced and compared phylogenetically based on a 320 nt sequence from the GC glycoprotein-encoding M segment. These results demonstrate that the HTNV isolates from NM-R are distinctly separated from HTNV isolated from the People’s Republic of China. These studies provide for improved disease risk assessments that identify military activities, rodent HTNV rates, and other factors associated with the transmission of hantaviruses during field training exercises.
The small intestines of 6 species of rodents and 1 species of insectivore were examined seasonally for Plagiorchis muris infection in 3 different localities in northern Gyeonggi-do (Province), near the demilitarized zone (DMZ). A total of 1,496 animals, including 1,366 Apodemus agrarius, 54 Crocidura lasiura (insectivore), 32 Mus musculus, 28 Micronytus fortis, 9 Eothenomys regulus, 6 Micronys minutus, and 3 Cricetulus triton, were live-trapped at Yeoncheon-gun (n = 351), Paju-shi (804) and Pocheon-gun (343) at 3-mo intervals from December 2004 to September 2005. A total of 1,647 P. muris were collected from 72 (5.3%) A. agrarius. The infection rate was the highest in Pocheon-gun (8.2%), followed by Yeoncheon-gun (5.0%) and Paju-shi (4.2%). A higher infection rate was observed in A. agrarius captured during September (19.4%) than those captured during December (3.0%), June (2.6%), or April (0%). However, the worm burden was the highest in June (av. 32.1/animal), followed by September (24.7), December (4.0), and April (0). None of the other animal species were found infected with P. muris. The results reveal that A. agrarius is a natural definitive host for P. muris, and infection rates and worm burdens vary seasonally and geographically.
Plagiorchis muris; wild rodent; Apodemus agrarius; prevalence; worm burden; Gyeonggi-do (Province)
Anaplasmosis, caused by Anaplasma phagocytophilum and Anaplasma platys, and ehrlichiosis, caused by Ehrlichia chaffeensis, Ehrlichia ewingii, the "Panola Mountain Ehrlichia" and Ehrlichia muris-like pathogens have been identified as emerging tick borne infectious diseases in dogs and human patients. Persistent intravascular infection with these bacteria is well documented in dogs, but is less well documented in human beings.
Serology and PCR targeting multiple microbial genes, followed by DNA sequencing, was used to test sequential blood samples. Tissue culture isolation was attempted in two laboratories.
A. platys, E. chaffeensis, and E. ewingii DNA was amplified from two Anaplasma and Ehrlichia seronegative family members and their dog, all lacking typical symptoms of anaplasmosis or ehrlichiosis. Following treatment with doxycycline, the dog and mother were Anaplasma and Ehrlichia spp. PCR negative.
Sequential PCR testing provided molecular evidence supporting intravascular persistence of A. platys and Ehrlichia spp. in two humans and their dog. Diagnosticians and clinicians should consider the potential for co-infections due to these tick borne organisms.
Anaplasma; Ehrlichia; Rickettsemia; PCR; DNA sequencing
Until recently, the single known exception to the rodent-hantavirus association was Thottapalayam virus (TPMV), a long-unclassified virus isolated from the Asian house shrew (Suncus murinus). Robust gene amplification techniques have now uncovered several genetically distinct hantaviruses from shrews in widely separated geographic regions. Here, we report the characterization of a newly identified hantavirus, designated Imjin virus (MJNV), isolated from the lung tissues of Ussuri white-toothed shrews of the species Crocidura lasiura (order Soricomorpha, family Soricidae, subfamily Crocidurinae) captured near the demilitarized zone in the Republic of Korea during 2004 and 2005. Seasonal trapping revealed the highest prevalence of MJNV infection during the autumn, with evidence of infected shrews' clustering in distinct foci. Also, marked male predominance among anti-MJNV immunoglobulin G antibody-positive Ussuri shrews was found, whereas the male-to-female ratio among seronegative Ussuri shrews was near 1. Plaque reduction neutralization tests showed no cross neutralization for MJNV and rodent-borne hantaviruses but one-way cross neutralization for MJNV and TPMV. The nucleotide and deduced amino acid sequences for the different MJNV genomic segments revealed nearly the same calculated distances from hantaviruses harbored by rodents in the subfamilies Murinae, Arvicolinae, Neotominae, and Sigmodontinae. Phylogenetic analyses of full-length S, M, and L segment sequences demonstrated that MJNV shared a common ancestry with TPMV and remained in a distinct out-group, suggesting early evolutionary divergence. Studies are in progress to determine if MJNV is pathogenic for humans.
A total of 1,496 rodents and insectivores were live-trapped at Yeoncheon-gun (n = 351), Paju-shi (804), and Pocheon-gun (343), Gyeonggi-do (Province), and examined for intestinal helminths, including Neodiplostomum seoulense, seasonally from December 2004 to September 2005. Six species of rodents, including Apodemus agrarius (1,366), Mus musculus (32), Micronytus fortis (28), Eothenomys regulus (9), Micronys minutus (6), and Cricetulus triton (3), and 1 species of insectivores Crocidura lasiura (54) were collected. A total of 321 adult N. seoulense were collected from 19 (1.4%) A. agrarius. The worm burden ranged from 1 to 101 per A. agrarius (mean; 16.9). No N. seoulense was observed in other rodent or insectivore species examined. The infection rate during autumn (4.5%) was higher than those during spring (0.8%), summer (0.8%), and winter (0.5%). The average number of N. seoulense in infected A. agrarius was the highest in spring (66.0 specimens), followed by autumn (15.2), winter (4.5), and summer (3.3). This study first confirms that A. agrarius is a natural definitive host for N. seoulense, and demonstrates that the infection rates and intensities vary seasonally and geographically.
Neodiplostomum seoulense; wild rodent; Apodemus agrarius; prevalence; worm burden; Gyeonggi-do
With the exception of Bartonella spp. or Cytauxzoon felis, feline vector-borne pathogens (FVBP) have been less frequently studied in North America and are generally under-appreciated as a clinical entity in cats, as compared to dogs or people. This study investigated selected FVBP seroreactivity and PCR prevalence in cats using archived samples.
Feline blood samples submitted to the Vector Borne Diseases Diagnostic Laboratory (VBDDL) at North Carolina State University College of Veterinary Medicine (NCSU-CVM) between 2008 and 2013 were tested using serological assays and PCR. An experimental SNAP® Multi-Analyte Assay (SNAP® M-A) (IDEXX Laboratories, Inc. Westbrook, Maine, USA) was used to screen all sera for antibodies to Anaplasma and Ehrlichia genus peptides and A.phagocytophilum, A.platys, B.burgdorferi, E.canis, E.chaffeensis, and E.ewingii species-specific peptides. PCR assays were used to amplify Anaplasma or Ehrlichia DNA from extracted ethylenediaminetetraacetic acid (EDTA)-anti-coagulated blood samples. Amplicons were sequenced to identify species.
Overall, 7.8 % (56/715) of cats were FVBP seroreactive and 3.2 % (13/406) contained Anaplasma or Ehrlichia DNA. Serologically, B.burgdorferi (5.5 %) was the most prevalent FVBP followed by A.phagocytophilum (1.8 %). Ehrlichia spp. antibodies were found in 0.14 % (12/715) of cats with species-specific seroreactivity to E.canis (n = 5), E.ewingii (n = 2) and E.chaffeensis (n = 1). Of seropositive cats, 16 % (9/56) were exposed to more than one FVBP, all of which were exposed to B.burgdorferi and either A.phagocytophilum (n = 7) or E.ewingii (n = 2). Based upon PCR and DNA sequencing, 4, 3, 3, 2, and 1 cat were infected with A.phagocytophilum, A.platys, E. ewingii, E. chaffeensis and E.canis, respectively.
Cats are exposed to and can be infected with vector-borne pathogens that commonly infect dogs and humans. To our knowledge, this study provides the first evidence for E.chaffeensis and E.ewingii infection in naturally-exposed cats in North America. Results from this study support the need for regional, serological and molecular FVBP prevalence studies, the need to further optimize serodiagnostic and PCR testing for cats, and the need for prospective studies to better characterize clinicopathological disease manifestations in cats infected with FVBP.
Anaplasma; Borrelia; Ehrlichia; Cats; Vector-borne pathogens
The prevalence and diversity of tick-borne zoonotic bacteria (Borrelia spp., Anaplasma phagocytophilum, Coxiella burnetii, and spotted fever group rickettsiae) infecting 253 small mammals captured in the Basque Country (Spain) were assessed using PCR and reverse line blot hybridization. Trapping sites were selected around sheep farms (study 1, 2000 to 2002) and recreational parks (study 2, 2003 to 2005). The majority of the studied mammals (162) were wood mice (Apodemus sylvaticus), but six other different species were also analyzed: yellow-necked mice (Apodemus flavicollis), shrews (Crocidura russula and Sorex coronatus), bank voles (Clethrionomys glareolus), domestic mice (Mus domesticus), and moles (Talpa europaea). The results showed an infection rate ranging from 10.7% to 68.8%, depending on the small mammal species. One C. russula shrew and one A. sylvaticus mouse gave positive reactions for A. phagocytophilum, and C. burnetii was detected in two domestic mice and one A. sylvaticus mouse in a farm. The DNA of Borrelia spp. was detected in 67 animals (26.5%), most of them presenting positive hybridization with the probe for Borrelia sp. strain R57, the new Borrelia species previously detected in small mammals in our region. Furthermore, a second PCR and reverse line blot hybridization specific for B. burgdorferi sensu lato revealed the presence of Borrelia afzelii in 6.3% of C. glareolus voles and 14.3% of S. coronatus shrews. All small mammals were negative for spotted fever group rickettsiae. These results highlight the relevance of small mammals as reservoirs of some zoonotic bacteria.
A total of 650 ticks, including 13 species from five genera, were collected from animals, from people, or by flagging of the vegetation at sites on the Thai-Myanmar border and in Vietnam. They were tested by PCR to detect DNA of bacteria of the order Rickettsiales. Three Anaplasma spp. were detected in ticks collected in Thailand, including (i) Anaplasma sp. strain AnDa465, which was considered a genotype of Anaplasma platys (formerly Ehrlichia platys) and which was obtained from Dermacentor auratus ticks collected from dogs; (ii) Anaplasma sp. strain AnAj360, which was obtained from Amblyomma javanense ticks collected on a pangolin; and (iii) Anaplasma sp. strain AnHl446, which was closely related to Anaplasma bovis and which was detected in Haemaphysalis lagrangei ticks collected from a bear. Three Ehrlichia spp. were identified, including (i) Ehrlichia sp. strain EBm52, which was obtained from Boophilus microplus ticks collected from cattle from Thailand; (ii) Ehrlichia sp. strain EHh324, which was closely related to Ehrlichia chaffeensis and which was detected in Haemaphysalis hystricis ticks collected from wild pigs in Vietnam; and (iii) Ehrlichia sp. strain EHh317, which was closely related to Ehrlichia sp. strain EBm52 and which was also detected in H. hystricis ticks collected from wild pigs in Vietnam. Two Rickettsia spp. were detected in Thailand, including (i) Rickettsia sp. strain RDla420, which was detected in Dermacentor auratus ticks collected from a bear, and (ii) Rickettsia sp. strain RDla440, which was identified from two pools of Dermacentor larvae collected from a wild pig nest. Finally, two bacteria named Eubacterium sp. strain Hw124 and Eubacterium sp. strain Hw191 were identified in Haemaphysalis wellingtoni ticks collected from chicken in Thailand; these strains could belong to a new group of bacteria.
Tanganya virus (TGNV), the only shrew-associated hantavirus reported to date from sub-Saharan Africa, is harbored by the Therese's shrew (Crocidura theresae), and is phylogenetically distinct from Thottapalayam virus (TPMV) in the Asian house shrew (Suncus murinus) and Imjin virus (MJNV) in the Ussuri white-toothed shrew (Crocidura lasiura). The existence of myriad soricid-borne hantaviruses in Eurasia and North America would predict the presence of additional hantaviruses in sub-Saharan Africa, where multiple shrew lineages have evolved and diversified.
Lung tissues, collected in RNAlater®, from 39 Buettikofer's shrews (Crocidura buettikoferi), 5 Jouvenet's shrews (Crocidura jouvenetae), 9 West African pygmy shrews (Crocidura obscurior) and 21 African giant shrews (Crocidura olivieri) captured in Côte d'Ivoire during 2009, were systematically examined for hantavirus RNA by RT-PCR.
A genetically distinct hantavirus, designated Azagny virus (AZGV), was detected in the West African pygmy shrew. Phylogenetic analysis of the S, M and L segments, using maximum-likelihood and Bayesian methods, under the GTR+I+Γ model of evolution, showed that AZGV shared a common ancestry with TGNV and was more closely related to hantaviruses harbored by soricine shrews than to TPMV and MJNV. That is, AZGV in the West African pygmy shrew, like TGNV in the Therese's shrew, did not form a monophyletic group with TPMV and MJNV, which were deeply divergent and basal to other rodent- and soricomorph-borne hantaviruses. Ancestral distributions of each hantavirus lineage, reconstructed using Mesquite 2.74, suggested that the common ancestor of all hantaviruses was most likely of Eurasian, not African, origin.
Genome-wide analysis of many more hantaviruses from sub-Saharan Africa are required to better understand how the biogeographic origin and radiation of African shrews might have contributed to, or have resulted from, the evolution of hantaviruses.
Rhipicephalus sanguineus is a ubiquitous tick responsible for transmitting Ehrlichia canis and most likely Anaplasma platys to dogs, as either single or co-infections. The objective of this study was to assess the effects of either simultaneous or sequential experimental infections with E. canis and A. platys on hematological and serological parameters, duration of infection, and efficacy of doxycycline therapy in dogs infected with one or both organisms. Six dogs per group were either uninfected, A. platys infected, E. canis infected, A. platys and E. canis co-infected, A. platys infected and E. canis challenged or E. canis infected and A. platys challenged at day 112 post-infection (PI). Doxycycline treatment was initiated at 211 days PI, followed by dexamethasone immunosuppression beginning 410 days PI.
Initially, transient decreases in hematocrit occurred in all groups infected with E. canis, but the mean hematocrit was significantly lower in the A. platys and E. canis co-infected group. All dogs except the controls developed marked thrombocytopenia after initial infection followed by gradually increased platelet counts by 112 days PI in groups with the single infections, while platelet counts remained significantly lower in the A. platys and E. canis co-infected group. Both sequential and simultaneous infections of A. platys and E. canis produced an enhanced humoral immune response to A. platys when compared to infection with A. platys alone. Likewise, co-infection with E. canis and A. platys resulted in a more persistent A. platys infection compared to dogs infected with A. platys only, but nearly all A. platys infected dogs became A. platys PCR negative prior to doxycycline treatment. E. canis infected dogs, whether single or co-infected, remained thrombocytopenic and E. canis PCR positive in blood for 420 days. When treated with doxycycline, all E. canis infected dogs became E. canis PCR negative and the thrombocytopenia resolved. Despite immunosuppression, neither A. platys nor E. canis DNA was PCR amplified from doxycycline-treated dogs.
The results of this study demonstrate that simultaneous or sequential infection with A. platys and E. canis can alter various pathophysiological parameters in experimentally infected dogs, and because natural exposure to multiple tick-borne pathogens occurs frequently in dogs, awareness of co-infection is important in clinical practice.
Leptospirosis is an acute, febrile disease occurring in humans and animals worldwide. Leptospira spp. are usually transmitted through direct or indirect contact with the urine of infected reservoir animals. Among wildlife species, rodents act as the most important reservoir for both human and animal infection. To gain a better understanding of the occurrence and distribution of pathogenic leptospires in rodent and shrew populations in Germany, kidney specimens of 2973 animals from 11 of the 16 federal states were examined by PCR. Rodent species captured included five murine species (family Muridae), six vole species (family Cricetidae) and six shrew species (family Soricidae). The most abundantly trapped animals were representatives of the rodent species Apodemus flavicollis, Clethrionomys glareolus and Microtus agrestis. Leptospiral DNA was amplified in 10% of all animals originating from eight of the 11 federal states. The highest carrier rate was found in Microtus spp. (13%), followed by Apodemus spp. (11%) and Clethrionomys spp. (6%). The most common Leptospira genomospecies determined by duplex PCR was L. kirschneri, followed by L. interrogans and L. borgpetersenii; all identified by single locus sequence typing (SLST). Representatives of the shrew species were also carriers of Leptospira spp. In 20% of Crocidura spp. and 6% of the Sorex spp. leptospiral DNA was detected. Here, only the pathogenic genomospecies L. kirschneri was identified.
Leptospira spp.; leptospirosis; rodents; shrews; Germany
Human granulocytic anaplasmosis (HGA) and human monocytic ehrlichiosis (HME) are emerging, tick-borne, zoonotic infectious diseases caused by Anaplasma phagocytophilum and Ehrlichia chaffeensis, respectively. Early diagnosis is essential for rapid clinical treatment to avoid misdiagnosis and severe patient outcomes. Simple, sensitive and reliable diagnostic methods are urgently needed. In this study, we developed a duplex real-time PCR assay targeting the A. phagocytophilum ankA gene and the E. chaffeensis TRP120 gene, respectively. The lowest limit of detection of the duplex real-time PCR assay was 100 copies of the targeted A. phagocytophilum ankA gene and the E. chaffeensis TRP120 gene per reaction, and the specificity was 100%. Detection in blood DNA samples from the acute stage of illness for 22 HGA cases and 8 HME cases indicated that the duplex real-time PCR assay was more sensitive than the nested PCR assay. The infection of Citellusundulatus Pallas with A. phagocytophilum and E. chaffeensis was first confirmed in Xinjiang Province and the positive rate was 3.1% for A. phagocytophilum, 6.3% for E. chaffeensis and 3.1% for co-infection with both pathogens. The rates of A. phagocytophilum and E. chaffeensis infection of D. silvarum ticks collected from Shanxi Province were 8.2% and 14.8%, respectively, and the co-infection rate was 3.3%. The rates of A. phagocytophilum and E. chaffeensis infection in H. longicornis ticks collected from Shandong Province were 1.6% and 6.3%, respectively, and the co-infection rate was 1.6%.
Biting midges (Culicoides: Ceratopogonidae) were collected by Mosquito Magnet® traps at the Neutral Nations Supervisory Commission (NNSC) camp and Daeseongdong village inside the demilitarized zone (DMZ) and near the military demarcation line (MDL) separating North and South Korea and at Warrior Base (US Army training site) and Tongilchon 3 km south of the DMZ in northern Gyeonggi Province, Republic of Korea (ROK), from May-October 2010-2012, to determine their seasonal distributions. A total of 18,647 Culicoides females (18,399; 98.7%) and males (248; 1.3%) comprising 16 species were collected. Overall, the most commonly collected species was Culicoides nipponensis (42.9%), followed by C. erairai (29.2%), C. punctatus (20.3%), C. arakawae (3.3%), C. pallidulus (1.8%), and C. circumscriptus (1.4%), while the remaining 10 species accounted for only 1.1% of all Culicoides spp. collected. The seasonal distribution of C. nipponensis was bimodal, with high numbers collected during May-June and again during September. C. erairai was more frequently collected during June-July, followed by sharply decreased populations from August-October. C. punctatus was collected in low numbers from May-September with high numbers collected during October. C. erairai was predominantly collected from the NNSC camp (85.1% of all C. erairai collected) located adjacent to the MDL at Panmunjeom in the northernmost part of Gyeonggi-do (Province), while other sites yielded low numbers of specimens.
Culicoides nipponensis; Culicoides erairai; Culicoides punctatus; biting midge; Korea
Ixodes spp. tick-borne zoonotic diseases are present across the Holarctic in humans, domestic animals, and wildlife. Small mammals are reservoirs for the rickettsial pathogen Anaplasma phagocytophilum and tick vectors may include catholic-feeding bridge vectors as well as host-specialist or nidicolous ticks. Far western North American communities in which A. phagocytophilum is maintained are complex ecologically, with multiple reservoir host and tick species, multiple strains of the bacterial pathogen A. phagocytophilum and differences in dynamics of hosts and vectors across heterogeneous landscapes. We evaluated sites in northern California in order to identify primarily nidicolous ticks and the hosts they infest. A total of 667 ticks was found in 11 study sites, including 288 on flags and 379 attached to small mammals. Larvae were over-represented among attached ticks and adults on flags. The most abundant species was I. pacificus. Two-hundred fourteen nidicolous ticks were found, most abundantly I. angustus and I. spinipalpis. All adult I. ochotonae, I. auritulus, I. angustus, I. jellisoni, and I. woodi were female, while the male:female ratio of I. spinipalpis was 1.2:1 and 1:1 for I. pacificus. The greatest number of ticks was obtained from Tamias ochrogenys, Peromyscus spp., and Neotoma fuscipes. Of 234 small mammal individuals that were infested with Ixodes spp., only 81 (34.6%) were infested with I. pacificus. The remaining infested small mammals hosted nidicolous tick species. Eight ticks were PCR-positive, including 6 I. pacificus (one adult, one larva, and 6 nymphs), and 2 adult I. ochotonae and high PCR prevalences of 18% and 9% were detected in woodrats and chipmunks, respectively. Nymphal I. angustus ticks were active year-long with a possible increase in August while larval activity was only observed in December and spring months and adults only during spring and fall. Overall, we show high tick species richness and year-round high levels of infestation on rodents by several different nidicolous ticks in areas where A. phagocytophilum is enzootic, including on reported reservoir species.
Anaplasma phagocytophilum; Granulocytic anaplasmosis; Ixodes angustus; Ixodes ochotonae; Ixodes pacificus
Rickettsia conorii is transmitted by Rhipicephalus sanguineus ticks and causes Mediterranean Spotted Fever (MSF) in humans. Although dogs are considered the natural host of the vector, the clinical and epidemiological significance of R. conorii infection in dogs remains unclear. The aim of this prospective study was to investigate whether Rickettsia infection causes febrile illness in dogs living in areas endemic for human MSF.
Dogs from southern Italy with acute fever (n = 99) were compared with case–control dogs with normal body temperatures (n = 72). Serology and real-time PCR were performed for Rickettsia spp., Ehrlichia canis, Anaplasma phagocytophilum/A. platys and Leishmania infantum. Conventional PCR was performed for Babesia spp. and Hepatozoon spp. Acute and convalescent antibodies to R. conorii, E. canis and A. phagocytophilum were determined.
The seroprevalence rates at first visit for R. conorii, E. canis, A. phagocytophilum and L. infantum were 44.8%, 48.5%, 37.8% and 17.6%, respectively. The seroconversion rates for R. conorii, E. canis and A. phagocytophilum were 20.7%, 14.3% and 8.8%, respectively. The molecular positive rates at first visit for Rickettsia spp., E. canis, A. phagocytophilum, A. platys, L. infantum, Babesia spp. and Hepatozoon spp. were 1.8%, 4.1%, 0%, 2.3%, 11.1%, 2.3% and 0.6%, respectively. Positive PCR for E. canis (7%), Rickettsia spp. (3%), Babesia spp. (4.0%) and Hepatozoon spp. (1.0%) were found only in febrile dogs. The DNA sequences obtained from Rickettsia and Babesia PCRs positive samples were 100% identical to the R. conorii and Babesia vogeli sequences in GenBank®, respectively. Febrile illness was statistically associated with acute and convalescent positive R. conorii antibodies, seroconversion to R. conorii, E. canis positive PCR, and positivity to any tick pathogen PCRs. Fourteen febrile dogs (31.8%) were diagnosed with Rickettsia spp. infection based on seroconversion and/or PCR while only six afebrile dogs (12.5%) seroconverted (P = 0.0248). The most common clinical findings of dogs with Rickettsia infection diagnosed by seroconversion and/or PCR were fever, myalgia, lameness, elevation of C-reactive protein, thrombocytopenia and hypoalbuminemia.
This study demonstrates acute febrile illness associated with Rickettsia infection in dogs living in endemic areas of human MSF based on seroconversion alone or in combination with PCR.
Dog; Febrile illness; Rickettsia conorii; Ehrlichia; Anaplasma; Babesia; Hepatozoon; Leishmania; Serology; PCR
In rural parts of Africa, dogs live in close association with humans and livestock, roam freely, and usually do not receive prophylactic measures. Thus, they are a source of infectious disease for humans and for wildlife such as protected carnivores. In 2011, an epidemiological study was carried out around three conservation areas in Uganda to detect the presence and determine the prevalence of vector-borne pathogens in rural dogs and associated ticks to evaluate the risk that these pathogens pose to humans and wildlife.
Serum samples (n = 105), blood smears (n = 43) and blood preserved on FTA cards (n = 38) and ticks (58 monospecific pools of Haemaphysalis leachi and Rhipicephalus praetextatus including 312 ticks from 52 dogs) were collected from dogs. Dog sera were tested by indirect immunofluorescence to detect the presence of antibodies against Rickettsia conorii and Ehrlichia canis. Antibodies against R. conorii were also examined by indirect enzyme immunoassay. Real time PCR for the detection of Rickettsia spp., Anaplasmataceae, Bartonella spp. and Babesia spp. was performed in DNA extracted from FTA cards and ticks.
99 % of the dogs were seropositive to Rickettsia spp. and 29.5 % to Ehrlichia spp. Molecular analyses revealed that 7.8 % of the blood samples were infected with Babesia rossi, and all were negative for Rickettsia spp. and Ehrlichia spp. Ticks were infected with Rickettsia sp. (18.9 %), including R. conorii and R. massiliae; Ehrlichia sp. (18.9 %), including E. chaffeensis and Anaplasma platys; and B. rossi (1.7 %). Bartonella spp. was not detected in any of the blood or tick samples.
This study confirms the presence of previously undetected vector-borne pathogens of humans and animals in East Africa. We recommend that dog owners in rural Uganda be advised to protect their animals against ectoparasites to prevent the transmission of pathogens to humans and wildlife.
Babesia; Dogs; East Africa; Ehrlichia; Rickettsia; Tick-borne pathogens
Borrelia burgdorferi, the causative agent of Lyme disease (LD), and Babesia, Bartonella, and Ehrlichia species (spp.) are recognized tick-borne pathogens in humans worldwide. Using serology and molecular testing, the incidence of these pathogens was investigated in symptomatic patients from Australia.
Sera were analyzed by an immunofluorescent antibody assay (IFA) followed by immunoglobulin (Ig)G and IgM Western blot (WB) assays. Both whole blood and sera were analyzed for detection of specific Borrelia spp. DNA using multiplex polymerase chain reaction (PCR) testing. Simultaneously, patients were tested for Babesia microti, Babesia duncani, Anaplasma phagocytophilum, Ehrlichia chaffeensis, and Bartonella henselae infection by IgG and IgM IFA serology, PCR, and fluorescent in situ hybridization (FISH).
Most patients reported symptom onset in Australia without recent overseas travel. 28 of 51 (55%) tested positive for LD. Of 41 patients tested for tick-borne coinfections, 13 (32%) were positive for Babesia spp. and nine (22%) were positive for Bartonella spp. Twenty-five patients were tested for Ehrlichia spp. and (16%) were positive for Anaplasma phagocytophilum while none were positive for Ehrlichia chaffeensis. Among the 51 patients tested for LD, 21 (41%) had evidence of more than one tick-borne infection. Positive tests for LD, Babesia duncani, Babesia microti, and Bartonella henselae were demonstrated in an individual who had never left the state of Queensland. Positive testing for these pathogens was found in three others whose movements were restricted to the east coast of Australia.
The study identified a much larger tick-borne disease (TBD) burden within the Australian community than hitherto reported. In particular, the first cases of endemic human Babesia and Bartonella disease in Australia with coexisting Borrelia infection are described, thus defining current hidden and unrecognized components of TBD and demonstrating local acquisition in patients who have never been abroad.
Borrelia; lyme disease; Babesia; Bartonella; ehrlichiosis; Australia; humans
A survey to determine the geographical distribution and relative abundance of potential vectors of scrub typhus was conducted from October to November 2006 at 13 localities throughout the Republic of Korea. Apodemus agrarius accounted for 97.6% (80/82) of all rodents, while only 2 Myodes regulus (2/82) were collected. A total of 10,860 chiggers were collected from A. agrarius belonging to 4 genera and 8 species, while only Walchia fragilis (40) was collected from Myodes regulus. Leptotrombidium pallidum (8,137; 74.9%), a vector of scrub typhus, was the predominant species collected from A. agrarius followed by Leptotrombidium scutellare (2,057, 18.9%), Leptotrombidium palpale (279; 2.7%), Leptotrombidium orientale (232; 2.1%), and Leptotrombidium zetum (79; 0.7%), Neotrombicula tamiyai (58; 0.5%), Euschoengastica koreaensis (16; 0.1%), and Cheladonta ikaoensis (2; < 0.1%). L. pallidum was the predominant chigger collected at collection sites in Gangwon (100%), Gyeonggi (87.2%), Chungnam (100%), Chungbuk (100%), Jeonbuk (73.9%), Jeonnam (77.0%), and Gyeongbuk (66.1%) provinces, whereas L. scutellare was the predominant chigger collected in Gyeongnam province (77.9%) and Jeju Island (62.3%). Data suggest a correlation between chigger population abundance and human cases of scrub typhus in Korea.
Leptotrombidium; chigger mites; geographical distribution; scrub typhus
Recently, Imjin virus (MJNV), a genetically distinct hantavirus, was isolated from lung tissues of the Ussuri white-toothed shrew (Crocidura lasiura) captured near the demilitarized zone in the Republic of Korea. To clarify the genetic diversity of MJNV, partial M- and L-segment sequences were amplified from lung tissues of 12 of 37 (32.4%) anti-MJNV IgG antibody-positive Ussuri white-toothed shrews captured between 2004 and 2010. A 531-nucleotide region of the M segment (coordinates 2,255 to 2,785) revealed that the 12 MJNV strains differed by 0-12.2% and 0-2.3% at the nucleotide and amino acid levels, respectively. A similar degree of nucleotide (0.2-11.9%) and amino acid (0-3.8%) difference was found in a 632-nucleotide length of the L segment (coordinates 962 to 1,593) of nine MJNV strains. Phylogenetic analyses, based on the partial M and L segments of MJNV strains generated by the neighbor-joining and maximum likelihood methods, showed geographic-specific clustering, akin to the phylogeography of rodent-borne hantaviruses.
Although tick-borne diseases are important causes of morbidity and mortality in dogs in tropical areas, there is little information on the agents causing these infections in the Caribbean.
We used PCRs to test blood from a cross-section of dogs on St Kitts for Ehrlichia (E.) canis, Babesia (B.) spp., Anaplasma (A.) spp. and Hepatozoon (H.) spp. Antibodies against E. canis and A. phagocytophilum/platys were detected using commercial immunochromatography tests. Records of the dogs were examined retrospectively to obtain clinical and laboratory data.
There was serological and/or PCR evidence of infections of dogs with E. canis (27%; 46/170), Babesia spp. (24%; 90/372) including B. canis vogeli (12%; 43/372) and B. gibsoni (10%; 36/372), A. platys (11%; 17/157) and H. canis (6%; 15/266). We could not identify the Babesia sp. detected in nine dogs. There was evidence of multiple infections with dual infections with E. canis and B. canis vogeli (8%; 14/179) or B. gibsoni (7%; 11/170) being the most common. There was agreement between immunochromatography and PCR test results for E. canis for 87% of dogs. Only 13% of exposed dogs had signs of a tick-borne disease and 38% had laboratory abnormalities. All 10 dogs presenting for a recheck after treatment of E. canis with doxycycline were apparently healthy although all remained seropositive and six still had laboratory abnormalities despite an average of two treatments with the most recent being around 12 months previously. Infections with Babesia spp. were also mainly subclinical with only 6% (4/67) showing clinical signs and 13% (9/67) having laboratory abnormalities. Similarly, animals with evidence of infections with A. platys and H. canis were largely apparently healthy with only occasional laboratory abnormalities.
Dogs are commonly infected with tick-borne pathogens in the Caribbean with most having no clinical signs or laboratory abnormalities.
Ixodes ricinus is a major vector for a range of microbial pathogens and the most prevalent and widely distributed tick species on the European continent, occurring in both natural and urban habitats. Nevertheless, little is known about the relative density of ticks in these two ecologically distinct habitats and the diversity of tick-borne pathogens that they carry.
We compared densities of questing I. ricinus nymphs and adults in urban and natural habitats in Central and Northeastern Poland, assessed the prevalence and rate of co-infection with A. phagocytophilum, Rickettsia, Ehrlichia and ‘Ca. Neoehrlichia spp.’ in ticks, and compared the diversity of tick-borne pathogens using molecular assays (PCR).
Of the 1325 adults and nymphs, 6.2% were infected with at least one pathogen, with 4.4%, 1.7% and less than 0.5% being positive for the DNA of Rickettsia spp., A. phagocytophilum, Ehrlichia spp. and Ca. N. mikurensis, respectively. Although tick abundance was higher in natural habitats, the prevalence of the majority of pathogens was higher in urban forested areas.
We conclude that: (i) zoonotic genetic variants of A. phagocytophilum are widely distributed in the Polish tick population, (ii) although the diversity of tick borne pathogens was higher in natural habitats, zoonotic species/strains were detected only in urban forests, (iii) and we provide the first description of Ca. N. mikurensis infections in ticks in Poland.
Ixodes ricinus; Tick; Anaplasma; Rickettsia; Ehrlichia; Neoehrlichia; Natural habitats; Urban habitats; Tick density; Prevalence