中国部分地区实验猕猴巴尔通体感染状况及其遗传特征

【目的】五日热巴尔通体(Bartonella quintana)由体虱在人群中传播,可引起多种人类疾病包括战壕热。为进一步搜集猕猴是五日热巴尔通体自然宿主的证据,本研究调查了国内4个地区实验用猕猴五日热巴尔通体的感染状况,对菌株遗传特征进行了分析。【方法】采集猕猴全血和血清样品各550份,用于菌株分离、核酸和血清IgG抗体检测。应用6个管家基因扩增及测序方法进行菌株鉴定、系统发育及核苷酸多态性分析;应用随机扩增多态性DNA标记(Random amplified polymorphic DNA,RAPD)技术分析不同宿主来源菌株RAPD指纹图谱差异;应用间接免疫荧光法(Indirect immunofluorescence assay,IFA)检测血清中抗五日热巴尔通体IgG抗体水平。【结果】从550只猕猴中分离到8株五日热巴尔通体菌株,带菌率为1.5%;直接PCR检测550份全血核酸的总感染率为8.2%。普通猕猴血清阳性率为19.0%,感染水平明显高于食蟹猕猴(5.6%)。五日热巴尔通体与汉赛巴尔通体RAPD指纹图谱的带型完全不同,猴源和人源五日热巴尔通体菌株Fuller带型基本一致。不同宿主来源菌株核苷酸多态性分析显示,猴源菌株之间差异小,其与人源菌株差异较大。【结论】中国猕猴五日热巴尔通体感染水平较高,普通猕猴自然感染率及抗体水平明显高于食蟹猕猴,猴源与人源菌株的基因型有明显差异。     

Multi-Locus Sequence Typing of a Geographically and Temporally Diverse Sample of the Highly Clonal Human Pathogen Bartonella quintana

Bartonella quintana is a re-emerging pathogen and the causative agent of a variety of disease manifestations in humans including trench fever. Various typing methods have been developed for B. quintana, but these tend to be limited by poor resolution and, in the case of gel-based methods, a lack of portability. Multilocus sequence typing (MLST) has been used to study the molecular epidemiology of a large number of pathogens, including B. henselae, a close relative of B. quintana. We developed a MLST scheme for B. quintana based on the 7 MLST loci employed for B. henselae with two additional loci to cover underrepresented regions of the B. quintana chromosome. A total of 16 B. quintana isolates spanning over 60 years and three continents were characterized. Allelic variation was detected in five of the nine loci. Although only 8/4270 (0.002%) of the nucleotide sites examined were variable over all loci, these polymorphisms resolved the 16 isolates into seven sequence types (STs). We also demonstrate that MLST can be applied on uncultured isolates by direct PCR from cardiac valve tissue, and suggest this method presents a promising approach for epidemiological studies in this highly clonal organism. Phylogenetic and clustering analyses suggest that two of the seven STs form a distinct lineage within the population.     

Acquisition and excretion of Bartonella quintana by the cat flea,Ctenocephalides felis felis

Bartonella quintana is transmitted by the infected faeces of body lice. Recently, this bacterium was detected in cat fleas (Ctenocephalides felis) and in two humans with chronic adenopathy whose only risk factor was contact with cat fleas. In this study, a total of 960 C. felis were divided into 12 groups (2 control groups and 10 infected groups) each containing 80 fleas. The fleas were fed B. quintana‐inoculated human blood at different dilutions (≈3.6 × 10⁴ ? 8.4 × 10⁹ bacteria) for 4 days via an artificial membrane. Subsequently, all flea groups were fed uninfected blood until day 13 postinfection (dpi). On day 3 pi, B. quintana was detected with two specific genes by quantitative PCR in 60–100% of randomly chosen fleas per dilution: 52% (26/50) in the infected fleas in Trial 1 and 90% (45/50) of the fleas in Trial 2. B. quintana was also identified by molecular and culture assays in flea faeces. The average number of B. quintana as determined by qPCR decreased until the 11th dpi and was absent in both trials at the 13th dpi. Bacteria were localized only in the flea gastrointestinal gut by specific immunohistochemistry. Our results indicate that cat fleas can acquire B. quintana by feeding and release viable organisms into their faeces. Therefore, fleas may play a role as vectors of trench fever or other clinical manifestations that are caused by B. quintana. However, the biological role of C. felis in the transmission of B. quintana under natural conditions is yet to be defined.     

Host dispersal shapes the population structure of a tick‐borne bacterial pathogen

Birds are hosts for several zoonotic pathogens. Because of their high mobility, especially of longdistance migrants, birds can disperse these pathogens, affecting their distribution and phylogeography. We focused on Borrelia burgdorferi sensu lato, which includes the causative agents of Lyme borreliosis, as an example for tick‐borne pathogens, to address the role of birds as propagation hosts of zoonotic agents at a large geographical scale. We collected ticks from passerine birds in 11 European countries. B. burgdorferi s.l. prevalence in Ixodes spp. was 37% and increased with latitude. The fieldfare Turdus pilaris and the blackbird T. merula carried ticks with the highest Borrelia prevalence (92 and 58%, respectively), whereas robin Erithacus rubecula ticks were the least infected (3.8%). Borrelia garinii was the most prevalent genospecies (61%), followed by B. valaisiana (24%), B. afzelii (9%), B. turdi (5%) and B. lusitaniae (0.5%). A novel Borrelia genospecies “Candidatus Borrelia aligera” was also detected. Multilocus sequence typing (MLST) analysis of B. garinii isolates together with the global collection of B. garinii genotypes obtained from the Borrelia MLST public database revealed that: (a) there was little overlap among genotypes from different continents, (b) there was no geographical structuring within Europe, and (c) there was no evident association pattern detectable among B. garinii genotypes from ticks feeding on birds, questing ticks or human isolates. These findings strengthen the hypothesis that the population structure and evolutionary biology of tick‐borne pathogens are shaped by their host associations and the movement patterns of these hosts.     

Complete Genome Sequence of Bartonella quintana,a Bacterium Isolated from Rhesus Macaques

Bartonella quintana is a re-emerging pathogen and the causative agent of a broad spectrum of disease manifestations in humans. The present study reports the complete genome of B. quintana strain RM_11, which was isolated from rhesus macaques.     

Complex Population Structure of Borrelia burgdorferi in Southeastern and South Central Canada as Revealed by Phylogeographic Analysis

Lyme disease, caused by the bacterium Borrelia burgdorferi sensu stricto, is an emerging zoonotic disease in Canada and is vectored by the blacklegged tick, Ixodes scapularis. Here we used Bayesian analyses of sequence types (STs), determined by multilocus sequence typing (MLST), to investigate the phylogeography of B. burgdorferi populations in southern Canada and the United States by analyzing MLST data from 564 B. burgdorferi-positive samples collected during surveillance. A total of 107 Canadian samples from field sites were characterized as part of this study, and these data were combined with existing MLST data for samples from the United States and Canada. Only 17% of STs were common between both countries, while 49% occurred only in the United States, and 34% occurred only in Canada. However, STs in southeastern Ontario and southwestern Quebec were typically identical to those in the northeastern United States, suggesting a recent introduction into this region from the United States. In contrast, STs in other locations in Canada (the Maritimes; Long Point, Ontario; and southeastern Manitoba) were frequently unique to those locations but were putative descendants of STs previously found in the United States. The picture in Canada is consistent with relatively recent introductions from multiple refugial populations in the United States. These data thus point to a geographic pattern of populations of B. burgdorferi in North America that may be more complex than simply comprising northeastern, midwestern, and Californian groups. We speculate that this reflects the complex ecology and spatial distribution of key reservoir hosts.     

DNA identification and characterization of Campylobacter jejuni and Campylobacter coli isolated from caecal samples of chickens in Grenada

Aims: To speciate Campylobacter strains from the caeca of chickens in Grenada using PCR and to evaluate DNA‐based typing methods for the characterization of these isolates. Methods and Results: Isolates were speciated with two multiplex PCR assays and were typed with flaA‐RFLP, pulsed‐field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Results confirmed that Campylobacter coli strains were more predominant than Campylobacter jejuni strains. From 56 isolates, 18 were misidentified using biochemical tests. PFGE typing gave the highest discriminatory power among the methods used (Simpson’s index of diversity, D = 0·9061). However, the combination of flaA‐RFLP, PFGE and MLST results gave the highest discrimination for subtyping of these isolates (D = 0·9857). A band position tolerance of 4% in Bio Numerics was the most appropriate for the analysis of this database. MLST profiles were generally concordant with PFGE and/or flaA‐RFLP types. Several isolates exhibited new MLST sequence types (STs), and 43 of the 49 Camp. coli strains belonged to the ST‐828 clonal complex. Conclusions: Campylobacter coli was the most prevalent species isolated from broilers and layers in Grenada, and a combination of restriction and sequence methods was most appropriate for the typing of Camp. coli isolates. Campylobacter coli STs clustered with described poultry‐associated Camp. coli STs by phylogenetic analysis. Significance and Impact of the Study: Further studies to understand the predominance of Camp. coli within Campylobacter spp. from chickens in Grenada may help elucidate the epidemiology of these pathogens in chickens.     

Single‐Nucleotide Polymorphisms Reveal Patterns of Allele Sharing Across the Species Boundary Between Rhesus (Macaca mulatta) and Cynomolgus (M. fascicularis) Macaques

Both phenotypic and genetic evidence for asymmetric hybridization between rhesus (Macaca mulatta) and cynomolgus (Macaca fascicularis) macaques has been observed in the region of Indochina where both species are sympatric. The large‐scale sharing of major histocompatibility complex (MHC) class II alleles between the two species in this region supports the hypothesis that genes, and especially genes involved in immune response, are being transferred across the species boundary. This differential introgression has important implications for the incorporation of cynomolgus macaques of unknown geographic origin in biomedical research protocols. Our study found that for 2,808 single‐nucleotide polymorphism (SNP) markers, the minor allele frequencies (MAF) and observed heterozygosity calculated from a sample of Vietnamese cynomolgus macaques was significantly different from those calculated from samples of both Chinese rhesus and Indonesian cynomolgus macaques. SNP alleles from Chinese rhesus macaques were overrepresented in a sample of Vietnamese cynomolgus macaques relative to their Indonesian conspecifics and located in genes functionally related to the primary immune system. These results suggest that Indochinese cynomolgus macaques represent a genetically and immunologically distinct entity from Indonesian cynomolgus macaques. Am. J. Primatol. 75:135‐144, 2013. © 2012 Wiley Periodicals, Inc.     

Comparative genetics of a highly divergent DRB microsatellite in different macaque species

The DRB region of the major histocompatibility complex (MHC) of cynomolgus and rhesus macaques is highly plastic, and extensive copy number variation together with allelic polymorphism makes it a challenging enterprise to design a typing protocol. All intact DRB genes in cynomolgus monkeys (Mafa) appear to possess a compound microsatellite, DRB-STR, in intron 2, which displays extensive length polymorphism. Therefore, this STR was studied in a large panel of animals, comprising pedigreed families as well. Sequencing analysis resulted in the detection of 60 Mafa-DRB exon 2 sequences that were unambiguously linked to the corresponding microsatellite. Its length is often allele specific and follows Mendelian segregation. In cynomolgus and rhesus macaques, the nucleotide composition of the DRB-STR is in concordance with the phylogeny of exon 2 sequences. As in humans and rhesus monkeys, this protocol detects specific combinations of different DRB-STR lengths that are unique for each haplotype. In the present panel, 22 Mafa-DRB region configurations could be defined, which exceeds the number detected in a comparable cohort of Indian rhesus macaques. The results suggest that, in cynomolgus monkeys, even more frequently than in rhesus macaques, new haplotypes are generated by recombination-like events. Although both macaque species are known to share several identical DRB exon 2 sequences, the lengths of the corresponding microsatellites often differ. Thus, this method allows not only fast and accurate DRB haplotyping but may also permit discrimination between highly related macaque species.     

Rhesus macaques (Macaca mulatta) are natural hosts of specific Staphylococcus aureus lineages

Currently, there is no animal model known that mimics natural nasal colonization by Staphylococcus aureus in humans. We investigated whether rhesus macaques are natural nasal carriers of S. aureus. Nasal swabs were taken from 731 macaques. S. aureus isolates were typed by pulsed-field gel electrophoresis (PFGE), spa repeat sequencing and multi-locus sequence typing (MLST), and compared with human strains. Furthermore, the isolates were characterized by several PCRs. Thirty-nine percent of 731 macaques were positive for S. aureus. In general, the macaque S. aureus isolates differed from human strains as they formed separate PFGE clusters, 50% of the isolates were untypeable by agr genotyping, 17 new spa types were identified, which all belonged to new sequence types (STs). Furthermore, 66% of macaque isolates were negative for all superantigen genes. To determine S. aureus nasal colonization, three nasal swabs from 48 duo-housed macaques were taken during a 5 month period. In addition, sera were analyzed for immunoglobulin G and A levels directed against 40 staphylococcal proteins using a bead-based flow cytometry technique. Nineteen percent of the animals were negative for S. aureus, and 17% were three times positive. S. aureus strains were easily exchanged between macaques. The antibody response was less pronounced in macaques compared to humans, and nasal carrier status was not associated with differences in serum anti-staphylococcal antibody levels. In conclusion, rhesus macaques are natural hosts of S. aureus, carrying host-specific lineages. Our data indicate that rhesus macaques are useful as an autologous model for studying S. aureus nasal colonization and infection prevention.     

Aerosol exposure to Zaire ebolavirus in three nonhuman primate species: differences in disease course and clinical pathology

There is little known concerning the disease caused by Zaire ebolavirus (ZEBOV) when inhaled, the likely route of exposure in a biological attack. Cynomolgus macaques, rhesus macaques, and African green monkeys were exposed to aerosolized ZEBOV to determine which species might be the most relevant model of the human disease. A petechial rash was noted on cynomolgus and rhesus macaques after fever onset but not on African green monkeys. Fever duration was shortest in rhesus macaques (62.7 ± 16.3 h) and longest in cynomolgus macaques (82.7 ± 22.3 h) and African green monkeys (88.4 ± 16.7 h). Virus was first detectable in the blood 3 days after challenge; the level of viremia was comparable among all three species. Hematological changes were noted in all three species, including decreases in lymphocyte and platelet counts. Increased blood coagulation times were most pronounced in African green monkeys. Clinical signs and time to death in all three species were comparable to what has been reported previously for each species after parenteral inoculation with ZEBOV. These data will be useful in selection of an animal model for efficacy studies.     

Characterization of 47 MHC class I sequences in Filipino cynomolgus macaques

Cynomolgus macaques (Macaca fascicularis) provide increasingly common models for infectious disease research. Several geographically distinct populations of these macaques from Southeast Asia and the Indian Ocean island of Mauritius are available for pathogenesis studies. Though host genetics may profoundly impact results of such studies, similarities and differences between populations are often overlooked. In this study we identified 47 full-length MHC class I nucleotide sequences in 16 cynomolgus macaques of Filipino origin. The majority of MHC class I sequences characterized (39 of 47) were unique to this regional population. However, we discovered eight sequences with perfect identity and six sequences with close similarity to previously defined MHC class I sequences from other macaque populations. We identified two ancestral MHC haplotypes that appear to be shared between Filipino and Mauritian cynomolgus macaques, notably a Mafa-B haplotype that has previously been shown to protect Mauritian cynomolgus macaques against challenge with a simian/human immunodeficiency virus, SHIV_89.6P. We also identified a Filipino cynomolgus macaque MHC class I sequence for which the predicted protein sequence differs from Mamu-B*17 by a single amino acid. This is important because Mamu-B*17 is strongly associated with protection against simian immunodeficiency virus (SIV) challenge in Indian rhesus macaques. These findings have implications for the evolutionary history of Filipino cynomolgus macaques as well as for the use of this model in SIV/SHIV research protocols. Kevin J. Campbell and Ann M. Detmer contributed equally to this work.     

Identification of Entamoeba polecki with Unique 18S rRNA Gene Sequences from Celebes Crested Macaques and Pigs in Tangkoko Nature Reserve,North Sulawesi,Indonesia

Unique species of macaques are distributed across Sulawesi Island, Indonesia, and the details of Entamoeba infections in these macaques are unknown. A total of 77 stool samples from Celebes crested macaques (Macaca nigra) and 14 stool samples from pigs were collected in Tangkoko Nature Reserve, North Sulawesi, and the prevalence of Entamoeba infection was examined by PCR. Entamoeba polecki was detected in 97% of the macaques and all of the pigs, but no other Entamoeba species were found. The nucleotide sequence of the 18S rRNA gene in E. polecki from M. nigra was unique and showed highest similarity with E. polecki subtype (ST) 4. This is the first case of identification of E. polecki ST4 from wild nonhuman primates. The sequence of the 18S rRNA gene in E. polecki from pigs was also unique and showed highest similarity with E. polecki ST1. These results suggest that the diversity of the 18S rRNA gene in E. polecki is associated with differences in host species and geographic localization, and that there has been no transmission of E. polecki between macaques and pigs in the study area.     

Revisiting the phylogeny of Wolbachia in Collembola

The endosymbiont Wolbachia has been detected in a few parthenogenetic collembolans sampled in Europe and America, including three species of Poduromorpha, two species of Entomobryomorpha, and two species of Neelipleona. Based on 16S rRNA and ftsZ gene sequences, most of the Wolbachia infecting parthenogenetic collembolans were characterized as members of supergroup E and showed concordant phylogeny with their hosts. However, the two neelipleonan symbionts form another unique group, indicating that Wolbachia has infected parthenogenetic collembolans multiple times. In this study, five parthenogenetic collembolan species were identified as hosts of Wolbachia, and four new Wolbachia strains were reported for four collembolan species sampled in China, respectively, including a neelipleonan strain from Megalothorax incertus (wMinc). Our results demonstrated that the Wolbachia multilocus sequence typing (MLST) system is superior to the 16S rRNA + ftsZ approach for phylogenetic analyses of collembolan Wolbachia. The MLST system assigned these Wolbachia of parthenogenetic collembolans to supergroup E as a unique clade, which included wMinc, supporting the monophyletic origin of Wolbachia in parthenogenetic collembolan species. Moreover, our data suggested supergroup E as one of the most divergent lineages in Wolbachia and revealed the discrepancy between the phylogenies of Wolbachia from parthenogenetic collembolans and their hosts, which may result from the high level of genetic divergence between collembolan Wolbachia, in association with the geographic differentiation of their hosts, or the possible horizontal transmission of Wolbachia between different collembolan species.     

Diversity and evolution of the Wolbachia endosymbionts of Bemisia (Hemiptera: Aleyrodidae) whiteflies

Wolbachia is the most prevalent symbiont described in arthropods to date. Wolbachia can manipulate host reproduction, provide nutrition to insect hosts and protect insect hosts from pathogenic viruses. So far, 13 supergroups of Wolbachia have been identified. The whitefly Bemisia tabaci is a complex containing more than 28 morphologically indistinguishable cryptic species. Some cryptic species of this complex are invasive. In this study, we report a comprehensive survey of Wolbachia in B. tabaci and its relative B. afer from 1658 insects representing 54 populations across 13 provinces of China and one state of Australia. Based on the results of PCR or sequencing of the 16S rRNA gene, the overall rates of Wolbachia infection were 79.6% and 0.96% in the indigenous and invasive Bemisia whiteflies, respectively. We detected a new Wolbachia supergroup by sequencing five molecular marker genes including 16S rRNA, groEL, gltA, hcpA, and fbpA genes. Data showed that many protein‐coding genes have limitations in detecting and classifying newly identified Wolbachia supergroups and thus raise a challenge to the known Wolbachia MLST standard analysis system. Besides, the other Wolbachia strains detected from whiteflies were clustered into supergroup B. Phylogenetic trees of whitefly mitochondrial cytochrome oxidase subunit I and Wolbachia multiple sequencing typing genes were not congruent. In addition, Wolbachia was also detected outside the special bacteriocytes in two cryptic species by fluorescence in situ hybridization, indicating the horizontal transmission of Wolbachia. Our results indicate that members of Wolbachia are far from well explored.     

The Trw Type IV Secretion System of Bartonella Mediates Host-Specific Adhesion to Erythrocytes

Bacterial pathogens typically infect only a limited range of hosts; however, the genetic mechanisms governing host-specificity are poorly understood. The α-proteobacterial genus Bartonella comprises 21 species that cause host-specific intraerythrocytic bacteremia as hallmark of infection in their respective mammalian reservoirs, including the human-specific pathogens Bartonella quintana and Bartonella bacilliformis that cause trench fever and Oroya fever, respectively. Here, we have identified bacterial factors that mediate host-specific erythrocyte colonization in the mammalian reservoirs. Using mouse-specific Bartonella birtlesii, human-specific Bartonella quintana, cat-specific Bartonella henselae and rat-specific Bartonella tribocorum, we established in vitro adhesion and invasion assays with isolated erythrocytes that fully reproduce the host-specificity of erythrocyte infection as observed in vivo. By signature-tagged mutagenesis of B. birtlesii and mutant selection in a mouse infection model we identified mutants impaired in establishing intraerythrocytic bacteremia. Among 45 abacteremic mutants, five failed to adhere to and invade mouse erythrocytes in vitro. The corresponding genes encode components of the type IV secretion system (T4SS) Trw, demonstrating that this virulence factor laterally acquired by the Bartonella lineage is directly involved in adherence to erythrocytes. Strikingly, ectopic expression of Trw of rat-specific B. tribocorum in cat-specific B. henselae or human-specific B. quintana expanded their host range for erythrocyte infection to rat, demonstrating that Trw mediates host-specific erythrocyte infection. A molecular evolutionary analysis of the trw locus further indicated that the variable, surface-located TrwL and TrwJ might represent the T4SS components that determine host-specificity of erythrocyte parasitism. In conclusion, we show that the laterally acquired Trw T4SS diversified in the Bartonella lineage to facilitate host-restricted adhesion to erythrocytes in a wide range of mammals.     

Molecular Evidence for Distinct Genotypes of Monkey B Virus (Herpesvirus Simiae) Which Are Related to the Macaque Host Species

Although monkey B virus (herpesvirus simiae; BV) is common in all macaque species, fatal human infections appear to be associated with exposure to rhesus macaques (Macaca mulatta), suggesting that BV isolates from rhesus monkeys may be more lethal to nonmacaques than are BV strains indigenous to other macaque species. To determine if significant differences that would support this supposition exist among BV isolates, we compared multiple BV strains isolated from rhesus, cynomolgus, pigtail, and Japanese macaques. Antigenic analyses indicated that while the isolates were very closely related to one another, there are some antigenic determinants that are specific to BV isolates from different macaque species. Restriction enzyme digest patterns of viral DNA revealed marked similarities between rhesus and Japanese macaque isolates, while pigtail and cynomolgus macaque isolates had distinctive cleavage patterns. To further compare genetic diversity among BV isolates, DNA sequences from two regions of the viral genome containing genes that are conserved (UL27 and US6) and variable (US4 and US5) among primate alphaherpesviruses, as well as from two noncoding intergenic regions, were determined. From these sequence data and a phylogenetic analysis of them it was evident that while all isolates were closely related strains of BV, there were three distinct genotypes. The three BV genotypes were directly related to the macaque species of origin and were composed of (i) isolates from rhesus and Japanese macaques, (ii) cynomolgus monkey isolates, and (iii) isolates from pigtail macaques. This study demonstrates the existence of different BV genotypes which are related to the macaque host species and thus provides a molecular basis for the possible existence of BV isolates which vary in their levels of pathogenicity for nonmacaque species.     

Molecular cloning and sequence analysis of interleukin 16 from nonhuman primates and from the mouse

 Interleukin 16 (IL-16) is synthesized as a 67 000 M _r precursor (pro-IL-16), but only a carboxy terminal part of 12 000–14 000 M _r is secreted by CD8(⁺) lymphocytes. This lymphokine binds to CD4 and has been shown to induce migration, affect the activation state of T cells, and inhibit immunodeficiency virus replication. It has been suggested that CD8(⁺) cell-derived soluble factors play a pivotal role in protecting natural-host nonhuman primates from developing immunodeficiency following SIV infection. In a first attempt to address this question, we cloned and sequenced the IL-16 cDNA from different primates. Here we report the pro-IL-16 sequence from chimpanzees, African green monkeys (AGM), rhesus macaques, and cynomolgus macaques. In order to compare and analyze structural motifs possibly involved in processing, intracellular targeting, or secretion, we extended our study to the New World monkeys saimiri and aotus and to the mouse. Alignments of deduced amino acids reveal that the human protein shares 99% similarity to that of chimpanzees, approximately 95% to rhesus, cynomolgus and AGM, about 90% to aotus and saimiri, and 77.5% to the mouse. Phylogenetic analyses revealed the expected evolutionary groupings. Received: 27 August 1997 / Revised: 7 October 1997     

Identification of MhcMafa-DRB Alleles in a Cohort of Cynomolgus Macaques of Vietnamese Origin

Cynomolgus macaques have been used widely to build a research model of infectious and chronic diseases, as well as in transplantation studies, where disease susceptibility and/or resistance are associated with the major histocompatibility complex (MHC). To better elucidate polymorphisms and genetic differences in the Mafa‐DRB locus, and facilitate the experimental use of cynomolgus macaques, we used pool screening combined with cloning and direct sequencing of polymerase chain reaction products to characterize MhcMafa‐DRB gene alleles in 153 Vietnamese cynomolgus macaques. We identified 30 Mafa‐DRB alleles belonging to 17 allelic lineages, including four novel sequences that had not been documented in earlier reports. The highest frequency allele was Mafa‐DRB*W27:04, which was present in 7 of 35 (20%) monkeys. The next most frequent alleles were Mafa‐DRB*3:07 and Mafa‐DRB*W7:01, which were detected in 5 of 35 (14.3%) and 4 of 35 (11.4%) of the monkeys, respectively. The high‐frequency alleles in this Vietnamese population may be high priority targets for additional characterization of immune functions. Only the DRB1*03 and DRB1*10 lineages were also present in humans, whereas the remaining alleles were monkey‐specific lineages. We found 25 variable sites by aligning the deduced amino acid sequences of 29 identified alleles. Evolutionary and population analyses based on these sequences showed that human, rhesus, and cynomolgus macaques share several Mhc‐DRB lineages and the shared polymorphisms in the DRB region may be attributable to the existence of interbreeding between rhesus and cynomolgus macaques. This information will promote the understanding of MHC diversity and polymorphism in cynomolgus macaques and increase the value of this species as a model for biomedical research. Am. J. Primatol. 74:958‐966, 2012. © 2012 Wiley Periodicals, Inc.     

Determination of Wolbachia diversity in butterflies from Western Ghats, India, by a multigene approach

Members of the genus Wolbachia are intracellular bacteria that are widespread in arthropods and establish diverse symbiotic associations with their hosts, ranging from mutualism to parasitism. Here we present the first detailed analyses of Wolbachia in butterflies from India with screening of 56 species. Twenty-nine species (52%) representing five families were positive for Wolbachia. This is the first report of Wolbachia infection in 27 of the 29 species; the other two were reported previously. This study also provides the first evidence of infection in the family Papilionidae. A striking diversity was observed among Wolbachia strains in butterfly hosts based on five multilocus sequence typing (MLST) genes, with 15 different sequence types (STs). Thirteen STs are new to the MLST database, whereas ST41 and ST125 were reported earlier. Some of the same host species from this study carried distinctly different Wolbachia strains, whereas the same or different butterfly hosts also harbored closely related Wolbachia strains. Butterfly-associated STs in the Indian sample originated by recombination and point mutation, further supporting the role of both processes in generating Wolbachia diversity. Recombination was detected only among the STs in this study and not in those from the MLST database. Most of the strains were remarkably similar in their wsp genotype, despite divergence in MLST. Only two wsp alleles were found among 25 individuals with complete hypervariable region (HVR) peptide profiles. Although both wsp and MLST show variability, MLST gives better separation between the strains. Completely different STs were characterized for the individuals sharing the same wsp alleles.