Phylogenetic characterization of two novel species of the genus Bifidobacterium: Bifidobacterium saimiriisciurei sp. nov. and Bifidobacterium platyrrhinorum sp. nov.

Three bifidobacterial Gram-stain-positive, non-spore forming and fructose-6-phosphate phosphoketolase-positive strains, SMA1T, SMB2 and SMA15T were isolated from the faeces of two adult males of the squirrel monkey (Saimiri sciureus). On the basis of 16S rRNA gene sequence similarities, the type strain of Bifidobacterium primatium DSM 100687T (99.3%; similarity) was the closest neighbour to strains SMA1T and SMB2, whereas the type strain of Bifidobacterium stellenboschense DSM 23968T (96.5%) was the closest neighbour to strain SMA15T. The average nucleotide identity (ANI) values of SMA1T and SAM15T with the closely related type strains were 93.7% and 88.1%, respectively. The in silico DNA‒DNA hybridization values with the closest neighbours were 53.1% and 36.9%, respectively. GC contents of strains SMA1T and SMA15T were 63.6 and 66.4 mol%, respectively. Based on the phylogenetic, genotypic and phenotypic data obtained, the strains SMA1T and SMA15T clearly represent two novel taxa within the genus Bifidobacterium for which the names Bifidobacterium saimiriisciurei sp. nov. (type strain SMA1T = BCRC 81223T = NBRC 114049T = DSM 106020T) and Bifidobacterium platyrrhinorum sp. nov. (type strain SMA15T = BCRC 81224T = NBRC 114051T = DSM 106029T) are proposed.

Characterization of the phylogenetic diversity of five novel species belonging to the genus Bifidobacterium: Bifidobacterium castoris sp. nov., Bifidobacterium callimiconis sp. nov., Bifidobacterium goeldii sp. nov., Bifidobacterium samirii sp. nov. and Bifidobacterium dolichotidis sp. nov.

Five Bifidobacterium strains, i.e. 2020BT, 2028BT, 2033BT, 2034BT and 2036BT, were isolated from European beaver (Castor fiber), Goeldi's marmoset (Callimicogoeldii), black-capped squirrel monkey (Saimiriboliviensissubsp. peruviensis) and Patagonian mara (Dolichotispatagonum). All of these isolates were shown to be Gram-positive, facultative anaerobic, d-fructose 6-phosphate phosphoketolase-positive, non-motile and non-sporulating. Phylogenetic analyses based on 16S rRNA gene sequences, multilocus sequences (including hsp60, rpoB, dnaJ, dnaG and clpC genes) and the core genome revealed that bifidobacterial strains 2020BT, 2028BT, 2033BT, 2034BT and 2036BT exhibit close phylogenetic relatedness to Bifidobacterium biavatii DSM 23969T, Bifidobacterium bifidum LMG 11041T, Bifidobacterium choerinum LMG 10510T, Bifidobacterium gallicum LMG 11596T, Bifidobacterium imperatoris LMG 30297T, Bifidobacterium italicum LMG 30187T and Bifidobacterium vansinderenii LMG 30126T, respectively. Further genotyping based on the genome sequence of the isolated strains combined with phenotypic analyses, clearly show that these strains are distinct from each of the type strains of the so far recognized Bifidobacterium species. Thus, Bifidobacterium castoris sp. nov. (2020BT=LMG 30937T=CCUG 72816T), Bifidobacterium callimiconis sp. nov. (2028BT=LMG 30938T=CCUG 72814T), Bifidobacterium samirii sp. nov. (2033BT=LMG 30940T=CCUG 72817T), Bifidobacterium goeldii sp. nov. (2034BT=LMG 30939T=CCUG 72815T) and Bifidobacterium dolichotidis sp. nov. (2036BT=LMG 30941T=CCUG 72818T) are proposed as novel Bifidobacterium species.

Detection of mycobacterial infection in non-human primates using the Xpert MTB/RIF molecular assay.

Tuberculosis is a major public health concern, and diagnostic strategies applied to animal populations are scarce. As part of ongoing efforts to control tuberculosis dissemination at our animal facility, two non-human primates (NHP, Saimiri sciureus) presenting cutaneous lesions were examined for mycobacterial infection. Both animals tested positive for acid-fast bacilli and Mycobacterium tuberculosis using a molecular assay (IS6110 PCR). Animals were euthanized and several samples were tested for M. tuberculosis using the Xpert MTB/RIF assay. Many samples were positive for M. tuberculosis and rifampicin resistance, and some produced mycobacterial growth. Oral swabs from cage mates were then tested with Xpert MTB/RIF, and the majority tested positive for M. tuberculosis and rifampicin resistance, and produced growth in culture. To our knowledge, this is the first report of multidrug-resistant mycobacterial infection in NHP. Additionally, our data shows that the Xpert MTB/RIF assay can be useful as a screening tool for tuberculosis infection in NHP.

Antibiotic resistance in Staphylococcus sp. isolated from the vaginal environment of squirrel monkeys (Saimiri spp.) bred ex situ.

BACKGROUND: Squirrel monkeys (Saimiri spp.) have been widely used as animal models; however, the occurrence of Staphylococcus sp in their vaginal microbiota remains to be described. METHODS: Samples were collected from 175 adult squirrel monkeys to isolate Staphylococcus sp and to test for susceptibility to a panel of nine antimicrobial agents. RESULTS: Isolates with characteristics of the genus Staphylococcus were detected in 95 of 175 samples. Coagulase-negative staphylococci (CoNS) were the most common (95.8%, 91/95) isolates. Resistance to antibiotics was observed in 47.3% (45/95) of isolates. Resistance to tetracycline was observed in 28.5% (26/91), chloramphenicol in 15.4% (14/91), and methicillin in 13.2% (12/91) of CoNS. Coagulase-positive staphylococci were resistant to tetracycline, erythromycin, and methicillin. CONCLUSIONS: The presence of Staphylococcus sp in vaginal samples obtained from squirrel monkeys suggests that these animals were in a carrier state. Furthermore, isolating strains resistant to methicillin reinforces the biosafety care of a colony.

Outbreak of pasteurellosis in captive Bolivian squirrel monkeys (Saimiri boliviensis).

In September 2012, five Bolivian squirrel monkeys housed in a zoological park died within sequential several days without obvious clinical signs. In a necrospy, one monkey presented swelling of the kidney with multifocal white nodules in the parenchyma, and other two had pulmonary congestion. Histopathologically, multifocal bacterial colonies of gram-negative coccobacillus were found in the sinusoid of the liver in all monkeys examined (Nos.1-4). Additionally, purulent pyelonephritis, pneumonia and disseminated small bacterial colonies in blood vessels were observed. Immunohistochemically, the bacterial colonies from two monkeys were positive for P. multocida capsular serotype D. Based on these findings, these monkeys were diagnosed as septicemia caused by acute P. multocida infection.

Pathological changes in captive monkeys with spontaneous yersiniosis due to infection by Yersinia enterocolitica serovar O8.

An outbreak of fatal yersiniosis due to infection with Yersinia enterocolitica serovar O8 is documented in two species of captive monkey. Five of 50 squirrel monkeys (Saimiri sciureus) and one of two agile gibbons (Hylobates agilis) died following several days of diarrhoea. Necropsy examination revealed necrotizing enterocolitis and multifocal necrosis or abscesses in various organs. Microscopically, these lesions comprised multifocal necrosis with bacterial colonies, neutrophils and accumulation of nuclear debris. Occasional lesions included macrophages and abscess formation. Immunohistochemically, the bacteria were identified as Y. enterocolitica O8. In addition, Y. enterocolitica serotype O8 was isolated from animal organs in pure culture. This is the first report of fatal cases of infection with Y. enterocolitica serovar O8 in animals.

Seroepidemiological survey of pathogenic Yersinia in breeding squirrel monkeys in Japan.

To investigate the prevalence of antibodies to pathogenic Yersinia in breeding squirrel monkeys, the serum samples of 252 squirrel monkeys from 9 zoological gardens in Japan were tested by ELISA using plasmid-encoded Yersinia outer membrane protein (Yops) as the antigen. The cutoff value was calculated by using the serum samples of the squirrel monkeys from Suriname, where no prevalence of pathogenic Yersinia have been reported. According to the cutoff value, 164 of 252 (65.1%) squirrel monkeys were considered positive against pathogenic Yersinia. These positive monkeys belonged to 8 of the 9 zoological gardens, and the percentage of the seropositive monkeys ranged from 22.2 to 89.4%. Furthermore, in one zoological garden, the positive rate of the squirrel monkeys which were over 1 year old (95.7%) was significantly higher than those which were under 1 year old (23.3%). These results suggested that pathogenic Yersinia is highly prevalent among breeding monkeys in Japan.

Rapid diagnosis and quantification of Francisella tularensis in organs of naturally infected common squirrel monkeys (Saimiri sciureus).

Francisella tularensis, a small Gram-negative facultative intracellular bacterium, is the causative agent of tularaemia, a severe zoonotic disease transmitted to humans mostly by vectors such as ticks, flies and mosquitoes. The disease is endemic in many parts of the northern hemisphere. Among animals, the most affected species belong to rodents and lagomorphs, in particular hares. However, in the recent years, many cases of tularaemia among small monkeys in zoos were reported. We have developed a real-time PCR that allows to quantify F. tularensis in tissue samples. Using this method, we identified the spleen and the kidney as the most heavily infected organ containing up to 400 F. tularensis bacteria per simian host cell in two common squirrel monkeys (Saimiri sciureus) from a zoo that died of tularaemia. In other organs such as the brain, F. tularensis was detected at much lower titres. The strain that caused the infection was identified as F. tularensis subsp. holarctica biovar I, which is susceptible to erythromycin. The high number of F. tularensis present in soft organs such as spleen, liver and kidney represents a high risk for persons handling such carcasses and explains the transmission of the disease to a pathologist during post-mortem analysis. Herein, we show that real-time PCR allows a reliable and rapid diagnosis of F. tularensis directly from tissue samples of infected animals, which is crucial in order to attempt accurate prophylactic measures, especially in cases where humans or other animals have been exposed to this highly contagious pathogen.

Staphylococcus simiae sp. nov., isolated from South American squirrel monkeys.

Eight coagulase-negative, oxidase-negative and novobiocin-susceptible staphylococcal strains were isolated from the gastrointestinal tracts of South American squirrel monkeys (Saimiri sciureus L.). These strains were differentiated from known staphylococcal species on the basis of 16S rRNA gene and hsp60 gene sequencing, and from the most closely related species by using DNA-DNA hybridization, ribotyping, whole-cell protein profiles and biotyping. Phylogenetic analysis based on 16S rRNA gene sequences confirmed that these strains are members of the Staphylococcus aureus species group (99% similarity) but are biochemically similar to Staphylococcus piscifermentans, from which they can be phenotypically distinguished by resistance to polymyxin B, acid production from D-mannitol, the inability to hydrolyse aesculin and DNA and the absence of alpha-glucosidase. On the basis of these analyses, a novel species of the genus Staphylococcus is described, for which the name Staphylococcus simiae sp. nov. is proposed, with CCM 7213(T) (=LMG 22723(T)) as the type strain.

Yersinia enterocolitica serovar O:8 infection in breeding monkeys in Japan.

In the period from December 2002 to January 2003, 5 of 50 squirrel monkeys (Saimiri sciureus) housed at a Zoological Garden in the Kanto region of Japan died following a few days' history of diarrhea. After this outbreak had ended in the squirrel monkeys, 1 of 2 dark-handed gibbons (Hylobates agilis) died in April of 2003, showing similar clinical signs. We examined the organs of 3 of the dead squirrel monkeys and of the dark-handed gibbon, and Yersinia enterocolitica serovar O:8, which is the most pathogenic serovar of Y. enterocolitica, was isolated. In order to determine the source and the transmission route of infection, 98 fecal samples (45 from squirrel monkeys, 20 from other monkeys of 18 different species, and 33 from black rats captured around the monkey houses) and 7 water samples were collected in the Zoological Garden, and were examined for the prevalence of Y. enterocolitica serovar O:8. Serovar O:8 was isolated from 21 of 65 monkeys (32.3%) and 5 of 33 (15.2%) black rats (Rattus rattus). Furthermore, we examined the 30 isolates using molecular typing methods, pulsed field gel electrophoresis (PFGE), ribotyping using the RiboPrinter system, and restriction endonuclease analysis of virulence plasmid DNA (REAP), and compared the isolates in this outbreak with Japanese O:8 isolates previously identified. Genotyping showed that almost all the isolates were identical, and the genotype of the isolates was highly similar to that from wild rodents captured in Niigata Prefecture. This is the first report of fatal cases of Y. enterocolitica serovar O:8 infection in monkeys anywhere in the world.

The putative haemobartonella that influences Plasmodium falciparum parasitaemia in squirrel monkeys is a haemotrophic mycoplasma.

Splenectomised squirrel monkeys (Saimiri sciureus) are increasingly being used as an experimental host for human malaria studies, notably for the assessment of candidate vaccines against Plasmodium falciparum blood-stage infection. Recently, S. sciureus monkeys in our primate-breeding colony were reported to be asymptomatic carriers of a putative Haemobartonella species. Patent haemobartonella infection is frequently activated following splenectomy, and may interfere with studies on the course of P. falciparum parasitaemia in these animals. Here, we show by 16S rRNA gene sequence analysis that this wall-less bacterium is not a rickettsia but, instead, is a haemotrophic mycoplasma. Haemotrophic mycoplasmas are a newly identified group of mycoplasmas that parasitise the surfaces of erythrocytes of a wide variety of vertebrate hosts.

In vitro characteristics of Yersinia pseudotuberculosis of nonhuman primate origin.

Six strains of serotypes 1 or 2 of Y. pseudotuberculosis were isolated from dead squirrel monkeys, a cotton-top tamarin and a marmoset hybrid. All strains harboured a 71.6 kb plasmid, all were totally oxacillin-resistant and partially resistant to cephalosporins. Biochemically, serotypes 1 and 2 differed from each other in their beta-galactosidase production in a nonfermenter system, whereas the lack of rhamnose, maltose, salicin and trehalose fermentation seemed to be attributable to technical causes.

Purification and biochemical characterization of squirrel monkey retrovirus-H produced in a human lymphoblastoid cell line.

We found a type D retrovirus in a human lymphoblastoid cell line of B-cell lineage. Molecular cloning and nucleotide sequencing of the provirus genome revealed that this virus was closely related to squirrel monkey retrovirus (SMRV), and we designated this virus as SMRV-H. To investigate the relationship between these retroviruses, SMRV-H was purified from the virus-producing cells, and its biochemical properties were characterized. The cell-adhesive virus particles were successfully separated from the cell by a brief trypsin treatment and purified by velocity sedimentation. The purification of the virus was confirmed by electron microscopy. Major gag protein of the virus is phosphorylated, and has a molecular weight of 34 kDa. The virion-associated reverse transcriptase prefers Mg2+ to Mn2+. These properties of SMRV-H were almost the same as those of SMRV.

The B allele of the NS gene of avian influenza viruses, but not the A allele, attenuates a human influenza A virus for squirrel monkeys.

The nonstructural (NS) genes of avian influenza A viruses have been divided into two groups on the basis of nucleotide sequence homology, which we have referred to here as alleles A and B. We sequenced the NS genes of eight additional avian influenza A viruses in order to define the differences between these two alleles more thoroughly. Four of the viruses had NS gene sequences which resembled that of A/FPV/Rostock/34 and belonged to allele A while the other four viruses had NS gene sequences more similar to that of A/Duck/Alberta/76 and belonged to allele B. There was approximately 90% sequence homology within alleles and 72% homology between alleles. As previously reported the NS genes of human influenza A viruses belong to allele A. We constructed single gene avian-human reassortant influenza A viruses containing an allele A or B NS gene segment from an avian influenza A virus and all other genes from a human influenza A virus and tested these reassortants for their ability to grow in the respiratory tract of a nonhuman primate. Reassortants containing an avian NS gene segment of allele B were significantly restricted in growth in the respiratory tract of squirrel monkeys while reassortants with an allele A NS gene segment were not. The divergent evolution of the B NS allele in birds may have resulted in gene products which do not function optimally in cooperation with genes from a human virus in viral replication in primate respiratory epithelium.

[Epidemiology of Klebsiella pneumoniae infections in 2 colonies of squirrel monkeys and lemurs]. / Epidémiologie des infections a Klebsiella pneumoniae dans deux élevages de singes-écureuils et de lémuriens.

After a short summary of the occurrence, sources, phenotypic characteristics, epidemiologic markers, virulence factors and pathogenicity of the 7 species of Klebsiella, the author reported (1) an infection due to K. pneumoniae K5 in a breed of squirrel monkey. These animals were suffering from sub-cutaneous abcesses (Pasteur Institute of Cayenne, French Guyana). (2) The second example refers a fatal infection due to K. pneumoniae K2 in a colony of lemurs, at Mulhouse zoological garden (East of France). Both animal colonies were protected against infection by the use of specific anti-K. pneumoniae capsular polysaccharide vaccine.

Natural occurrence of black-pigmented Bacteroides species in the gingival crevice of the squirrel monkey.

The objective of this study was to determine whether the squirrel monkey (Saimiri scuireus) is indigenously colonized with black-pigmented bacteroides (BPB) resembling human Bacteroides gingivalis and Bacteroides intermedius (suspected periodontal pathogens) and to determine the usefulness of the squirrel monkey as an in vivo model for studying colonization by putative pathogens. We assayed the subgingival plaques of 138 monkeys of various ages and in four different colonies for the presence of anaerobic BPB microorganisms. We also tested half the animals for the presence of Actinobacillus actinomycetemcomitans. Clinical indices and levels of serum antibody to B. gingivalis were recorded. We detected BPB in 50% of the animals and A. actinomycetemcomitans in 69% of the animals. The presence of BPB was generally associated with increased age, increased gingival index, presence of calculus, and increased levels of serum antibody. These data indicate that the squirrel monkey may be a good model for studying the parameters of natural infection of the gingival crevice with suspected periodontopathogenic BPB microorganisms.

Detection of antibodies to Herpesvirus saimiri late antigens in human sera.

One hundred fifty sera from handlers of squirrel monkeys and 100 sera from individuals who had never handled monkeys were tested by immunofluorescence for antibodies reactive to structural proteins of Herpesvirus saimiri (HVS). Eleven (7.3%) of the occupationally exposed group and 4 (4%) of the noncontact group were seropositive for HVS by immunofluorescence assay, and 10 of these 15 (6.7 and 2%, respectively) were also seropositive for either the major glycoprotein (140 kD) or the major capsid protein (160 kD) of HVS by radioimmunoprecipitation assay. Two sera from handlers of squirrel monkeys, however, recognized many different HVS structural antigens by immunoprecipitation, and it seems unlikely that they could also be cross-reactive antibodies. Since these two sera did not contain antibodies to HVS early antigens or to the nonstructural antigens present in infected owl monkey kidney cells, and follow-up sera collected from the same individuals several months later were negative for antibodies to HVS, these individuals do not appear to have been infected by the virus. The risk that HVS poses to humans appears to be very low.

Characterization by anti-Ig monoclonal antibodies of protective and non-protective antibodies against asexual forms of Plasmodium falciparum in the Saimiri monkey.

Four monoclonal antibodies (mAb) which recognize distinct epitopes on Saimiri immunoglobulins were successfully used to characterize the protective and non-protective antibodies developed by this experimental host in response to infection by the human malaria parasite Plasmodium falciparum. Two of these mAb, 3F11/G10 and 3E4/H8, were IgG-specific and directed against conformationally conserved epitopes on the intact molecule. mAb 3A2/G6 and 4G3/B5 were specific for epitopes on two distinct L chain types of all Ig. Radioimmunoassay and immunoblots indicated that L-chains defined by 3A2/G6 were present in IgG molecules of type 3F11/G10, while L chains defined by 4G3/B5 were associated with IgG of type 3E4/H8. The use of these four mAb as immunoadsorbents allowed the purification of two IgG and two Ig populations. When assayed in vivo by passive transfer experiments in recipient monkeys previously infected by P. falciparum, protection could be associated with the IgG or Ig populations of type 3F11/G10-3A2/G6. In contrast, recipient animals which received the IgG type 3E4/H8 or Ig type 4G3/B5 presented rapidly evolving parasitemia similar to that in animals which received non-immune IgG or protective immune sera depleted of the above Ig or IgG fractions. The identification of protective and non-protective Ig populations will help in the evaluation of vaccine candidates against P. falciparum.

Nucleotide sequence analysis of squirrel monkey retrovirus reveals a novel primer-binding site for tRNALys1,2.

Nucleotide sequences of a DNA fragment containing the long terminal repeat (LTR) of squirrel monkey retrovirus (SMRV) were determined. Sequence analysis showed that the SMRV LTR is 456 base pairs (bp) long and is bounded by 2-bp inverted repeats. Within the U3 region, there are two 43-bp repeats and two 42-bp repeats which are homologous to each other. These repeats are likely to provide enhancer activities commonly observed in other enhancer sequences. Following the repeats are transcriptional regulatory sequences including a CAT box, a Goldberg-Hogness box, and a polyadenylation signal, all positioned within the U3 region of SMRV LTR. A 22-nucleotide sequence immediately downstream from the LTR was found to be complementary to tRNALys1,2, suggesting that tRNALys1,2 serves as the primer for the reverse transcription of SMRV viral RNA.