Latin American countries produce more than a quarter of the world’s beef and are a major global supplier of livestock protein. Tick-borne diseases (TBDs) are a major constraint to the livestock industry worldwide, including in Latin America. The aim of this study was to detect and characterise tick-borne pathogens in cattle from Santa Cruz, Bolivia, where no detailed epidemiological data are available. Blood samples were collected from 104 cattle. Apicomplexan parasites were detected by nested PCR amplification of the 18S ribosomal RNA gene (rDNA), and Anaplasmataceae was screened by the PCR amplification of 16S rDNA, followed by characterisation based on the heat shock protein and citrate synthase gene sequences. Babesia infection was observed in nine cattle (one Babesia bovis and eight Babesia bigemina), while Anaplasmataceae infection was detected in thirty-two cattle. A sequencing analysis confirmed the presence of Anaplasma marginale and Anaplasma platys-like. These results provide the first molecular evidence for the four above-mentioned tick-borne pathogens in cattle in Bolivia. This information improves our understanding of the epidemiology of TBDs and will help in formulating appropriate and improved pathogen control strategies.
The genus Flavivirus includes a range of mosquito-specific viruses in addition to well-known medically important arboviruses. Isolation and comprehensive genomic analyses of viruses in mosquitoes collected in Bolivia resulted in the identification of three novel flavivirus species. Psorophora flavivirus (PSFV) was isolated from Psorophora albigenu . The coding sequence of the PSFV polyprotein shares 60 % identity with that of the Aedes -associated lineage II insect-specific flavivirus (ISF), Marisma virus. Isolated PSFV replicates in both Aedes albopictus - and Aedes aegypti -derived cells, but not in mammalian Vero or BHK-21 cell lines. Two other flaviviruses, Ochlerotatus scapularis flavivirus (OSFV) and Mansonia flavivirus (MAFV), which were identified from Ochlerotatus scapularis and Mansonia titillans, respectively, group with the classical lineage I ISFs. The protein coding sequences of these viruses share only 60 and 40 % identity with the most closely related of known lineage I ISFs, including Xishuangbanna aedes flavivirus and Sabethes flavivirus, respectively. Phylogenetic analysis suggests that MAFV is clearly distinct from the groups of the current known Culicinae -associated lineage I ISFs. Interestingly, the predicted amino acid sequence of the MAFV capsid protein is approximately two times longer than that of any of the other known flaviviruses. Our results indicate that flaviviruses with distinct features can be found at the edge of the Bolivian Amazon basin at sites that are also home to dense populations of human-biting mosquitoes.
Infectious agents were isolated from the spleens of three wild mice (Apodemus argenteus) by intraperitoneal inoculation of the spleen homogenate into laboratory mice. The laboratory mice developed clinical signs and splenomegaly,andthreeisolatesweremaintainedbypassageinmice.Tetracyclineswereeffectiveinpreventing infection of mice with these agents, but streptomycin and penicillin were ineffective. The agents did not grow in bacterial growth media or chicken embryos. In smears of blood from infected mice stained by the Giemsa or the indirect immunofluorescence method, numerous organisms were found on the surfaces of erythrocytes. Electron microscopy revealed cell wall-less pleomorphic cocci of 350 to 700 nm in diameter. On the basis of theseresults,theisolateswereidentifiedasHaemobartonellamuris.Therewasnoantigeniccross-reactivitywith Rickettsia or Ehrlichia spp. or other related organisms. Western immunoblot analysis of three strains of H. muris with mouse antisera to H. muris revealed identical major antigens of 118, 65, 53, 45, and 40 kDa. By heteroduplexanalysisofthethreePCR-amplifiedsegmentsofthe16SrRNAgenes,thethreestrainsofH.muris were found to be identical. The 16S rRNA genes of one of theH. murisstrains, four strains ofH. felis, and two strains ofEperythrozoon suiswere sequenced and compared. The sequences of two strains ofH. felisfrom cats in California were identical, as were the sequences of a strain from a cat in Ohio and a strain from a cat in Florida,butthesimilarityofsequencesbetweentheCaliforniaandtheOhio-Floridastrainswasonly85%.The sequence of anH. murisstrain was unique and was more closely related to that of the Ohio-Florida strain of H.felis(89%)thantothatoftheCaliforniastrainofH.felis(84%).ThesequenceofE.suisfromapiginIllinois was identical to that from another pig from Taiwan. The similarity of the 16S rRNA gene sequence ofE. suis with those of threeHaemobartonellastrains was 84 to 92%, with that ofE. suisbeing most similar to that of the H. felis strain from California. In the phylogenetic analysis based on 16S rRNA gene sequences, the Haemobartonellaspp.andE.suisformedadistinctclademorecloselyrelatedtoMycoplasmaspp.(79to83%similarity) than toAnaplasma marginale(72 to 75% similarity). Our results suggest that theHaemobartonellaspp. andE. suismay be reclassified in the same genus in the familyMycoplasmataceae.
Anaplasma phagocytophilum, which belongs to the order Rickettsiales, is an obligate intracellular bacterium and causes an emerging, tickborne, and febrile infectious disease, anaplasmosis, in humans and other mammals. This bacterium expresses a variety of 44-kDa immunodominant proteins encoded by the p44/msp2 multigene family on the surface for the purpose of avoiding the host immune defense due to the antigenic variation. In Japan, little is known about the molecular and biological features of A. phagocytophilum. In this study, we tried to characterize in detail the p44/msp2 multigene family of A. phagocytophilum from two tick species, Ixodes persulcatus and I. ovatus in Japan. A total of 174 amino acid sequences from the recombinant p44/msp2 clones after TA cloning of the amplicons obtained from the ticks were phylogenetically analyzed. The results showed that most of the clone sequences from I. ovatus were very similar to each other, but the sequences from I. persulcatus were diverse, and the sequences from the ticks were distinct from those from a wild deer that was previously reported. These findings suggest that Ixodes ticks are probably responsible for the transmission of certain genetic variants of A. phagocytophilum and that additional organism selection might occur in I. ovatus.
SUMMARY: We surveyed β-lactamase-producing Escherichia coli from farm animals (chickens, pigs, and cattle) and raw retail meat in Shizuoka Prefecture, Japan. In total 305 E. coli isolates, 15 isolates collected from broilers, beef cattle, chicken meat, and pork meat, were found to contain β-lactamase genes encoding CTX-M-2, CTX-M-14, CMY-2, SHV-2, and/or TEM-1, whereas 7 possessed mutations in the ampC promoter region. The findings suggest that broilers are more important than other farm animals with regards to the surveillance of β-lactamase-producing E. coli in this region. Enterobacteriaceae that harbor β-lactamases, especially extended-spectrum β-lactamase (ESBL), are a significant pathogen in nosocomial infections and have become an international topic of concern in both farm animals and humans (1–4). Numerous hypotheses have suggested that resistant bacteria and/or resistant genes could spread between farm animals and humans. In Japan, ESBL- and class C βlactamase-producing Escherichia coli, Klebsiella spp., and Proteus mirabilis are often detected in humans (5–9). In farm animals, Shiraki et al. (10) first reported the isolation of CTXM-2-producing E. coli from cattle in Japan. Kojima et al. (11) reported the isolation of CTX-M-type-producing E. coli from broilers but not from cattle or pigs. Moreover, there has only been one report regarding the antibiotic resistance of E. coli isolates from raw chicken meat (12). The distribution of β-lactamase-producing E. coli in farm animals and food products therefore remains unclear in Japan. In this study we surveyed β-lactamase-producing E. coli in food-producing animals and raw retail meat obtained from farms and markets located in Shizuoka Prefecture in Japan. Livestock feces (from broilers, layers, pigs, cows, and beef cattle) were collected in farms, and raw meat (chicken, pork, and beef) was obtained from supermarkets or meat markets in Shizuoka Prefecture in the period 2004–2006. The fecal samples were placed onto DHL agar (Nissui, Tokoy, Japan) plates and incubated aerobically at 37°C overnight to allow E. coli growth. Meat samples (25 g of each) were suspended in Bacto buffered peptone water (Difco, Detroit, Mich., USA) and incubated at 37°C for 18 h. The cultures were then spread onto Chromocult Coliform Agar ES (Merck, Darmstadt, Germany) plates and further incubated at 37°C for 24 h. The antimicrobial susceptibilities of the E. coli isolates were determined by the broth microdilution method using Dry Plate Eiken (Eiken, Tokyo, Japan). The Clinical Laboratory Standards Institute (CLSI) guidelines were followed with regard to inoculum standardization, medium and incubation conditions, and the selection of internal qualitycontrol organisms (E. coli ATCC 25922). All E. coli isolates from the samples were tested for resistance against cefotaxime (CTX), ampicillin (ABPC), gentamicin (GM), tetracycline (TC), chloramphenicol (CP), ciprofloxacin (CPFX), fosfomycin (FOM), and imipenem (IPM). Potential ESBLproducing E. coli with low susceptibility to CTX (MIC ≥2 μg/ ml) were selected and a phenotypic confirmatory test was conducted using CTX and ceftazidime, with or without clavulanic acid, according to the CLSI recommendations. The blaTEM, blaSHV, blaCTX-M-1, blaCTX-M-2, blaCTX-M-9, blaPSE-1,
There is an urgent need for structurally novel anti-norovirus agents. In this study, we describe the synthesis, anti-norovirus activity, and structure–activity relationship (SAR) of a series of heterocyclic carboxamide derivatives. Heterocyclic carboxamide 1 (50% effective concentration (EC50)=37 µM) was identified by our screening campaign using the cytopathic effect reduction assay. Initial SAR studies suggested the importance of halogen substituents on the heterocyclic scaffold and identified 3,5-di-boromo-thiophene derivative 2j (EC50=24 µM) and 4,6-di-fluoro-benzothiazole derivative 3j (EC50=5.6 µM) as more potent inhibitors than 1. Moreover, their hybrid compound, 3,5-di-bromo-thiophen-4,6-di-fluoro-benzothiazole 4b, showed the most potent anti-norovirus activity with a EC50 value of 0.53 µM (70-fold more potent than 1). Further investigation suggested that 4b might inhibit intracellular viral replication or the late stage of viral infection.
Here, we describe for the first time the prevalence and genetic properties of Bartonella organisms in wild rodents in Japan. We captured 685 wild rodents throughout Japan (in 12 prefectures) and successfully isolated Bartonella organisms from 176 of the 685 rodents (isolation rate, 25.7%). Those Bartonella isolates were all obtained from the rodents captured in suburban areas (rate, 51.8%), but no organism was isolated from the animals captured in city areas. Sequence analysis of rpoB and gltA revealed that the Bartonella isolates obtained were classified into eight genetic groups, comprising isolates closely related to B. grahamii (A-I group), B. tribocorum and B. elizabethae (B-J group), B. tribocorum and B. rattimassiliensis (C-K group), B. rattimassiliensis (D-L group), B. phoceensis (F-N group), B. taylorii (G-O group), and probably two additional novel Bartonella species groups (E-M and H-P). B. grahamii, which is one of the potential causative agents of human neuroretinitis, was found to be predominant in Japanese rodents. In terms of the relationships between these Bartonella genetic groups and their rodent species, (i) the A-I, E-M, and H-P groups appear to be associated with Apodemus speciosus and Apodemus argenteus; (ii) the C-K, D-L, and F-N groups are likely implicated in Rattus rattus; (iii) the B-J group seems to be involved in Apodemus mice and R. rattus; and (iv) the G-O group is probably associated with A. speciosus and Clethrionomys voles. Furthermore, dual infections with two different genetic groups of bartonellae were found in A. speciosus and R. rattus. These findings suggest that the rodent in Japan might serve as a reservoir of zoonotic Bartonella infection.
Anaplasma phagocytophilum is an obligate intracellular bacterium and causes a febrile illness in humans and livestock. In nature, this bacterium is sustained in a tick-mammal cycle. Several p44/msp2-related genes are expressed from a single expression locus by gene conversion. In this study, we obtained 119 cDNA sequences of p44/msp2 transcripts from A. phagocytophilum in 6 Haemaphysalis ticks and 3 wild sika deer (Cervus nippon) in Japan. These 119 sequences were classified into 36 different variant sequences based on their similarities. The 36 cDNA sequences were phylogenetically grouped into 2 major clusters--tick- and deer-associated. The tick-associated sequences were further classified into 4 distinct subclusters, suggesting that A. phagocytophilum in ticks seems to selectively express specific p44/msp2 transcripts, such as the transcripts in the 4 subclusters that were closely related to previously identified p44/msp2 genes. The deer-associated sequences were also grouped into 4 subclusters, but these transcripts were probably more diverse than the transcripts derived from ticks. This might be due to the relatively nonselective expression of p44/msp2 in deer or the strain differences in A. phagocytophilum from ticks and deer in separate geographic regions or both. Thus, this study may contribute to the understanding of A. phagocytophilum p44/msp2 expression in nature in Japan.
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