American Journal of Primatology 76:103–110 (2014)
RESEARCH ARTICLE Nasopharyngeal Colonization by Potentially Pathogenic Bacteria Found in Healthy Semi‐Captive Wild‐Born Chimpanzees in Uganda LAWRENCE MUGISHA1,2*, SOPHIE KÖNDGEN3, DEOGRATIAS KADDU‐MULINDWA4, LYNNE GAFFIKIN5, 3 AND FABIAN H. LEENDERTZ 1 EcoHealth Research Group, Conservation & Ecosystem Health Alliance (CEHA), Kampala, Uganda 2 College of Veterinary Medicine, Animal Resources and Biosecurity, Makerere University, Kampala, Uganda 3 Research Group Emerging Zoonoses, Robert Koch‐Institute, Berlin, Germany 4 College of Health Sciences, Makerere University, Kampala, Uganda 5 Evaluation and Research Technologies for Health (EARTH) Inc., Woodside, California
Information on the chimpanzee nasopharygeal colonization in captive sanctuaries and in the wild is rare. This study was undertaken to establish the nasopharygeal colonization and potential bacterial pathogens in sanctuary chimpanzees as a basis for improving chimpanzee and employee health. Nasopharygeal colonization of 39 healthy chimpanzees were analyzed by microbiological cultivation method and polymerase chain reaction (PCR) targeting the bacterial 16S rRNA gene. We report four major phyla dominated by Proteobacteria (50%), Fermicutes (35.7%), Bacteriodes (7.1%), and Cynobacteria (7.1%) in healthy semi‐captive chimpanzees. Further classification based on 7‐base oligomers revealed the following genera: Streptococcus, Veillonella, Neisseria, Prevotella, Kingella and unclassified Cynobacteria, Actinobacillus, Bacteriodes and Pasteurellaceae. On microbiological cultivation we were able to identify and characterize some of the bacteria to species level as Klebsiella pneumonie and Pseudomonas aeruginosa being dominant bacteria with 54.7% and 50% colonization, respectively. Of these, Streptococcus, Neisseria, Klebsiella, and Haemophillus have representatives known to potentially cause severe respiratory disease. Our data present important information on chimpanzee nasopharygeal colonization as a guide to understanding disease processes and pharmaceutical therapies required for improving the health of chimpanzees. The results from this study will guide the processes to improve procedures for routine management of sanctuary chimpanzees and use it as a basis for evaluation of future reintroduction possibilities. Am. J. Primatol. 76:103–110, 2014. © 2013 Wiley Periodicals, Inc. Key words:
chimpanzees; nasophrygeal colonization; employee health; disease transmission; reintroduction
INTRODUCTION Habituating apes to human presence for close observations by researchers and tourists and growing numbers of primate sanctuaries have created new opportunities to expose non‐human primates to new disease agents. Bacterial diseases of human origin have been identified in great ape populations believed to play a major role in progression of infectious diseases especially respiratory diseases [Köndgen et al., 2008]. Recently we found high prevalence of drug‐resistance human Staphylococcus aureus in sanctuary chimpanzees in Uganda and Zambia [Schaumburg et al., 2012] and Central Africa [Nagel et al., 2012] providing evidence for transmission of human pathogenic bacteria to chimpanzees and vice‐versa. In addition, high resistance rates to antibiotics were found in both staff and chimpanzees but higher in chimpanzees to penicillin, oxacillin, tetracyclin, trimethoprim/sulfamethoxazol, erythro-
© 2013 Wiley Periodicals, Inc.
mycin and clindamycin [Schaumburg et al., 2012]. This is attributed to indiscriminate use of antibiotic in sanctuaries and or transmission of resistant isolates from caregivers to chimpanzees during routine care [Schaumburg et al., 2012]. Previously gorillas and chimpanzees in Uganda have been shown to be infected with human bacterial strains Contract grant sponsor: Robert Koch Institute, Berlin; contract grant sponsor: DAAD Postdoctoral Fellowship
Correspondence to: Lawrence Mugisha, EcoHealth Research Group, Conservation & Ecosystem Health Alliance (CEHA), P.O. Box 34153, Kampala, Uganda. E‐mail: [email protected]
Received 2 April 2013; revised 30 July 2013; revision accepted 9 August 2013 DOI: 10.1002/ajp.22212 Published online 15 October 2013 in Wiley Online Library (wileyonlinelibrary.com).
104 / Mugisha et al.
[Goldberg et al., 2008; Nizeyi et al., 2001; Rwego et al., 2009]. Virulent Streptococcus pneumonie clones were found in deceased chimpanzees and implicated in the recorded chimpanzee mortalities in the Tai Forest of Cote d’Ivoire [Chi et al., 2007]. Lethal pneumonia due to human respiratory syncytial virus (HRSV) and infection with S. pneumoniae was demonstrated as cause of death in a captive juvenile chimpanzee [Szentiks et al., 2009]. HRSV and human meta‐pneumovirus (HMPV) have been documented as cause of mortalities in different habituated gorilla and chimpanzee communities [Kaur et al., 2008; Köndgen et al., 2008; Williams et al., 2008]. Respiratory conditions associated with bronchopneumonia is a major recorded illness in sanctuaries (data not published) with mortalities in affected groups and are believed to be of human origin. Although respiratory diseases have been frequently observed in captive and also wild great apes [Chi et al., 2007; Goodall, 1983, 1986; Köndgen et al., 2008], no publications on the nasopharyngeal bacteria are found. A better understanding of the normal bacterial flora of chimpanzees and changes in flora over time in sanctuaries would help elucidate conditions that facilitate the introduction and/or proliferation of pathogenic bacteria flora. The nasopharynx as a major source of secretions containing bacteria plays an important role in both the development of disease and the spread of pathogens from one individual to another. In addition, bacterial flora in humans is known to play a role in nutrition, carcinogesis and resistance to colonization by other pathogens [Mackowiak, 1982]. Recent review of human oral microbiome identified 1,179 taxa at the species level in 13 phyla deposited in the human oral microbiome database (HOMD) (www.homd.org) providing a tool for use in the understanding the role of the microbiome in health and disease [Dewhirst et al., 2010]. More recently, high diversity of saliva microbiome in Batwa Pygmies has been documented with 40 microbial genera that had not previously described in human oral cavity [Nasidze et al., 2011]. Such kind of information and database is critically lacking in apes currently faced with the same disease challenges. Despite the multitude of contacts presenting opportunities for trans‐species disease transmission at the NICS and other sanctuaries across Africa as shown by the recent study of Schaumburg et al. [2012], risk specifics are not well known or documented. This underscores the need for and importance of epidemiological studies to measure the magnitude and direction of disease transmission risk as the basis for (i) improved employee health programming and (ii) re‐introduction into the wild from sanctuaries as a viable conservation strategy. Towards this end, this study was undertaken to document which bacterial pathogens are part of the
Am. J. Primatol.
natural flora of wild caught/rescued chimpanzee, using modern diagnostic tools. METHODS Ngamba Island Chimpanzee Sanctuary (NICS) was established in 1998 and currently cares for 42 rescued orphan chimpanzees on Ngamba Island. Ngamba Island (S 000 06/E 32°39, 0.46 km2, 1,160 m above sea level) is 100 ha of secondary rain forest and lies 23 km off Entebbe in the north‐west of Lake Victoria, and is part of the Koome group of islands, in the Mukono District, Uganda. By the time of sample collection, the group consisted of 23 females and 19 males with age group categorized as follows: 4 Infants (1–5years), 6 Juveniles (6–8 years), 12 sub‐adults (9– 11 years) and adults (12 years and more). They are managed semi‐intensively with supplementary feeding twice a day using domestically purchased fruits and vegetables. Specimen Collection Sterile oral swabs were collected from 39 chimpanzees of Ngamba Island Chimpanzee Sanctuary (NIC) during the annual health checks under anesthesia. None of the chimpanzee was ill at time of sample collection and 2 months prior to sample collection. The oral swabs were preserved in RNALater and stored at 80°C till the time of examination. Other nasopharyngeal swabs were taken from all chimpanzees and placed in Stewart’s transport medium immediately and transported to the Medical Microbiology Laboratory of the Medical School of Makerere University the same day within a few hours for culturing. The research study was approved by Uganda Wildlife Authority (UWA) and Uganda National Council of Science and Technology (UNCST). Non‐invasive samples were collected following the ethical and animal welfare standards according to Pan African Sanctuaries Alliance (PASA), International Primatological Society and American Society of Primatologists principals for ethical treatment of primates. The results of this study have been shared with sanctuaries through PASA Veterinary HealthCare workshops that then share information with respective governments. Bacterial Culturing Quality controlled media of Chocolate agar, 5% sheep blood agar and MacConkey agar with crystal violet plates were used in the study. These were inoculated with the nasopharyngeal samples and incubated at 35–37°C in 5% CO2 incubator except MacConkey which was incubated in an incubator at 35–37°C for 18–24 hr. On Chocolate agar plate a disk of Oleondomycin was placed to enable the growth of any Haemophilus spp. visible. After incubation the
Chimpanzee Nasopharyngeal Colonization / 105
plates were evaluated for growth and suspected colonies were subjected to identification. The gram‐ negative rods, after establishing a pure culture were identified using either API20E or API20NE (bioMerieux, France). Colonies of suspected S. pneumoniae were subjected to optochin test and bile solubility test.
fragments were purified by use of the QIAquick PCR purification kit (Qiagen). The purified PCR products from clones were sequenced and analyzed on an ABI Prism 3100 Genetic Analyzer [Sanger et al., 1977]. Sequence information obtained from the bacteria screenings were compared to sequences of the public databases for identification of the different species.
DNA Extraction
Phylogenetic Analysis
Nucleic acids DNA/RNA were extracted from oral swabs of 39 chimpanzees using QIAamp Viral RNA Min Kit (Qiagen®, QIAamp®, Hilden, Germany). Double extraction of DNA using the spin column protocol was used following manufactures instructions. The eluted DNA was stored in 1.5 Eppendorf tubes at 80°C till use for the next step.
Initial editing and alignment of amplified sequences were performed using SeqMan (www.dnastar.com). Edited sequences were analyzed using Ribosomal Database Project (RDP) analysis tools available online at http://rdp.cme.msu.edu [Cole et al., 2007; Maidak et al., 2001]. Thirty‐seven 16S rRNA gene sequences obtained from colony amplicons were first uploaded into SeqMatch version 3 that searches for its nearest neighbors and thereafter aligned and classified by myRDP analysis tool. 148,277 sequences were included in the search that is based on 7‐base oligomers. The data set was optimized for both type and non‐type strains, both environmental (uncultured) sequences and isolates, near full sequences (>1,200 bases) and of good quality sequences. Results from query sequences under bacteria were analyzed and those sequences classified to genus level recorded. The sequences were classified using RDP Classifier, a Naïve Bayesian Classifier for rapid assignment of rRNA sequences into the New Bacterial Taxonomy in Bergey’s Taxonomic Outline of Prokaryotes [Wang et al., 2007]. Each sequence was labeled with a set of taxa, from the domain to genus. The major taxonomic ranks are: domain, phylum, class, order, family, genus and species with occasional intermediate ranks such as “subclass” and “suborder.” SeqMatch, myRDP and/or public RDP sequences were used to create a phylogenetic tree using the Weighbor weighted neighbor‐joining tree building algorithm [Bruno et al., 2000; Cole et al., 2007]. The tree includes results from a bootstrap test using 100 replicates. Bootstraps higher than 50% are highlighted. The integrity of the amplified sequences displaying 99% identical. These include Haemophilus influenza, Neisseria menengitides, S. pnuemoniae, Kingella kingae, Nicotiana tabacum, Veillonella oral clone, and Provetella sp. Oral clone (Fig. 1). In addition, we analyzed the same samples using bacteriological culture methods and found that the oral cavity of the wild‐born captives chimpanzees in a sanctuary are colonized by both normal and potential
pathogenic bacteria with >50% harboring Klebsiella pneumoniae and Pseudomonas auruginosa (Table II). Further analysis using specific TaqMan PCR for S. pneumoniae performed on 38 chimpanzee oral samples revealed that 78.9% (30/38) chimpanzees were positive for S. pneumoniae. DISCUSSION Provision of proper care, restoration of health and socio‐psycho well‐being of newly rescued and traumatized orphaned apes is a priority management intervention in great ape sanctuaries. Frequent outbreaks of the respiratory related illnesses presents a big challenge to the management and care of ape communities especially newly arrived individuals. In this study, we analyzed the nasopharyngeal colonization of chimpanzees on Ngamba Island and found chimpanzee nasal pharynx colonized by four major phyla: Proteobacteria, Fermicutes, Bacteriodes, and Cynobacteria. Seventeen (45.9%) out of 37 amplified 16S rRNA gene clones could not be identified to genus level and were classified as unclassified Pasteurellaceae, Bacteriodales, and Lactobacillales. This group represents uncharacterized sequences 1,200 nucleotides in length within the RDP database. This is expected; as little or no work has been done on bacterial flora of chimpanzees and these sequences may be different bacterial species not cultivated and characterized previously. This data constitutes the nasopharyngeal colonization of chimpanzees previously unknown. Bacteriological cultivation method revealed more information bacterial species (gram positive and negative) colonizing the oral cavity of chimpanzees dominated by K. pneumonie (54.7%) and P. aeruginosa (50%; Table I) While TaqMan PCR, revealed that 78.9% (30/38) chimpanzee’s oral cavity to be
TABLE I. Bacterial Culture Results From 42 Wild‐Born Semi‐Captive Chimpanzees on Ngamba Island
Cultured bacteria
Positive chimpanzees
Gram stain
Pseudomonas aeruginosa
21 (50%)
Negative rods
Klebsiella pneumonie Bacillus Escherichia coli Staphylococcus aureus
23 12 17 3
Neg. coccobacilli Positive rods Negative rods Positive cocci
(54.7%) (28.5%) (40.5%) (7.1%)
Streptococcus pyogenes Proteus mirabilis
1(2.4%9 2 (4.7%)
Positive cocci Negative bacillus
Enterobacter aerogenes Other un‐identified bacteria
4 (9.5%)
Negative rods Both neg. cocci and rods
Am. J. Primatol.
Colony morphology description Large, green pyocanin pigment with irregular spreading margins and fruit like odour Mucoid, raised, thick, and watery Large, flat, white, serrated edge, and glistening Small, mucoid, entire, raised, Blue colonies on ECA, Round, glistening, smooth, opaque with golden pigmentation Round to ovoid cocci in chains/pairs Small rod‐like cells with characteristic concentric rings of growth Round smooth, convex white rods Pink colonies to mucoid on ECA
Chimpanzee Nasopharyngeal Colonization / 107
Fig. 1. Phylogenetic analysis of bacteria diversity detected in the oral cavity of healthy semi‐captive chimpanzees using 16S rRNA analysis. The color‐coded names of chimpanzees followed by accession number of bacteria spp. was used in branches. This tree was rooted by using an Actinomyces denticolens (T) 16S rRNA sequence as an out‐group (Accession no. NCTC 11490). The scale bar represents an expected nucleotide substitution rate of 1%.
colonized by S. pneumoniae. Interestingly, some of the identified bacterial species or phylotypes by all methods are known potential respiratory pathogens in humans (K. pneumoniae, S. pnuemoniae, H. influenza and Neisseria meningitides). The study shows that the nasopharynx of chimpanzees is colonized by both non‐pathogenic and pathogenic bacteria. The nasopharyngeal carriage of potential pathogenic bacteria in healthy semi‐captive chimpanzees is likely to be influenced by close contact with
caregivers and the diet that is composed of domestic fruits and vegetables purchased in local markets. This colonization presents risk factors to the development of respiratory diseases in chimpanzees especially in periods of stress. Our results are similar with documented human studies where human nasopharynx has been shown to be colonized by potential respiratory pathogens H. influenza, S. pnuemoniae and Moraxella catarrhalis in early childhood [Barbosa‐Cesnik et al., 2006; Darboe
Am. J. Primatol.
108 / Mugisha et al.
TABLE II. Classification of 28 Isolates Sequences From Oral Swabs of Four Chimpanzees Using Naïve Bayesian rRNA Classifier Version 1.0 No. 100% similarity of Classified bacterial isolates to genus level Chimp name
Streptococcus
Neisseria
Veillonella
Kingella
Asega
4
—
2
—
Okech
1
2
—
—
3 8 28.5
3 2 7 25.0
2 7.1
1 1 3.6
Kisembo Umutama Total isolates % of isolates
Unclassified bacteria group
RESEARCH ARTICLE Nasopharyngeal Colonization by Potentially Pathogenic Bacteria Found in Healthy Semi‐Captive Wild‐Born Chimpanzees in Uganda LAWRENCE MUGISHA1,2*, SOPHIE KÖNDGEN3, DEOGRATIAS KADDU‐MULINDWA4, LYNNE GAFFIKIN5, 3 AND FABIAN H. LEENDERTZ 1 EcoHealth Research Group, Conservation & Ecosystem Health Alliance (CEHA), Kampala, Uganda 2 College of Veterinary Medicine, Animal Resources and Biosecurity, Makerere University, Kampala, Uganda 3 Research Group Emerging Zoonoses, Robert Koch‐Institute, Berlin, Germany 4 College of Health Sciences, Makerere University, Kampala, Uganda 5 Evaluation and Research Technologies for Health (EARTH) Inc., Woodside, California
Information on the chimpanzee nasopharygeal colonization in captive sanctuaries and in the wild is rare. This study was undertaken to establish the nasopharygeal colonization and potential bacterial pathogens in sanctuary chimpanzees as a basis for improving chimpanzee and employee health. Nasopharygeal colonization of 39 healthy chimpanzees were analyzed by microbiological cultivation method and polymerase chain reaction (PCR) targeting the bacterial 16S rRNA gene. We report four major phyla dominated by Proteobacteria (50%), Fermicutes (35.7%), Bacteriodes (7.1%), and Cynobacteria (7.1%) in healthy semi‐captive chimpanzees. Further classification based on 7‐base oligomers revealed the following genera: Streptococcus, Veillonella, Neisseria, Prevotella, Kingella and unclassified Cynobacteria, Actinobacillus, Bacteriodes and Pasteurellaceae. On microbiological cultivation we were able to identify and characterize some of the bacteria to species level as Klebsiella pneumonie and Pseudomonas aeruginosa being dominant bacteria with 54.7% and 50% colonization, respectively. Of these, Streptococcus, Neisseria, Klebsiella, and Haemophillus have representatives known to potentially cause severe respiratory disease. Our data present important information on chimpanzee nasopharygeal colonization as a guide to understanding disease processes and pharmaceutical therapies required for improving the health of chimpanzees. The results from this study will guide the processes to improve procedures for routine management of sanctuary chimpanzees and use it as a basis for evaluation of future reintroduction possibilities. Am. J. Primatol. 76:103–110, 2014. © 2013 Wiley Periodicals, Inc. Key words:
chimpanzees; nasophrygeal colonization; employee health; disease transmission; reintroduction
INTRODUCTION Habituating apes to human presence for close observations by researchers and tourists and growing numbers of primate sanctuaries have created new opportunities to expose non‐human primates to new disease agents. Bacterial diseases of human origin have been identified in great ape populations believed to play a major role in progression of infectious diseases especially respiratory diseases [Köndgen et al., 2008]. Recently we found high prevalence of drug‐resistance human Staphylococcus aureus in sanctuary chimpanzees in Uganda and Zambia [Schaumburg et al., 2012] and Central Africa [Nagel et al., 2012] providing evidence for transmission of human pathogenic bacteria to chimpanzees and vice‐versa. In addition, high resistance rates to antibiotics were found in both staff and chimpanzees but higher in chimpanzees to penicillin, oxacillin, tetracyclin, trimethoprim/sulfamethoxazol, erythro-
© 2013 Wiley Periodicals, Inc.
mycin and clindamycin [Schaumburg et al., 2012]. This is attributed to indiscriminate use of antibiotic in sanctuaries and or transmission of resistant isolates from caregivers to chimpanzees during routine care [Schaumburg et al., 2012]. Previously gorillas and chimpanzees in Uganda have been shown to be infected with human bacterial strains Contract grant sponsor: Robert Koch Institute, Berlin; contract grant sponsor: DAAD Postdoctoral Fellowship
Correspondence to: Lawrence Mugisha, EcoHealth Research Group, Conservation & Ecosystem Health Alliance (CEHA), P.O. Box 34153, Kampala, Uganda. E‐mail: [email protected]
Received 2 April 2013; revised 30 July 2013; revision accepted 9 August 2013 DOI: 10.1002/ajp.22212 Published online 15 October 2013 in Wiley Online Library (wileyonlinelibrary.com).
104 / Mugisha et al.
[Goldberg et al., 2008; Nizeyi et al., 2001; Rwego et al., 2009]. Virulent Streptococcus pneumonie clones were found in deceased chimpanzees and implicated in the recorded chimpanzee mortalities in the Tai Forest of Cote d’Ivoire [Chi et al., 2007]. Lethal pneumonia due to human respiratory syncytial virus (HRSV) and infection with S. pneumoniae was demonstrated as cause of death in a captive juvenile chimpanzee [Szentiks et al., 2009]. HRSV and human meta‐pneumovirus (HMPV) have been documented as cause of mortalities in different habituated gorilla and chimpanzee communities [Kaur et al., 2008; Köndgen et al., 2008; Williams et al., 2008]. Respiratory conditions associated with bronchopneumonia is a major recorded illness in sanctuaries (data not published) with mortalities in affected groups and are believed to be of human origin. Although respiratory diseases have been frequently observed in captive and also wild great apes [Chi et al., 2007; Goodall, 1983, 1986; Köndgen et al., 2008], no publications on the nasopharyngeal bacteria are found. A better understanding of the normal bacterial flora of chimpanzees and changes in flora over time in sanctuaries would help elucidate conditions that facilitate the introduction and/or proliferation of pathogenic bacteria flora. The nasopharynx as a major source of secretions containing bacteria plays an important role in both the development of disease and the spread of pathogens from one individual to another. In addition, bacterial flora in humans is known to play a role in nutrition, carcinogesis and resistance to colonization by other pathogens [Mackowiak, 1982]. Recent review of human oral microbiome identified 1,179 taxa at the species level in 13 phyla deposited in the human oral microbiome database (HOMD) (www.homd.org) providing a tool for use in the understanding the role of the microbiome in health and disease [Dewhirst et al., 2010]. More recently, high diversity of saliva microbiome in Batwa Pygmies has been documented with 40 microbial genera that had not previously described in human oral cavity [Nasidze et al., 2011]. Such kind of information and database is critically lacking in apes currently faced with the same disease challenges. Despite the multitude of contacts presenting opportunities for trans‐species disease transmission at the NICS and other sanctuaries across Africa as shown by the recent study of Schaumburg et al. [2012], risk specifics are not well known or documented. This underscores the need for and importance of epidemiological studies to measure the magnitude and direction of disease transmission risk as the basis for (i) improved employee health programming and (ii) re‐introduction into the wild from sanctuaries as a viable conservation strategy. Towards this end, this study was undertaken to document which bacterial pathogens are part of the
Am. J. Primatol.
natural flora of wild caught/rescued chimpanzee, using modern diagnostic tools. METHODS Ngamba Island Chimpanzee Sanctuary (NICS) was established in 1998 and currently cares for 42 rescued orphan chimpanzees on Ngamba Island. Ngamba Island (S 000 06/E 32°39, 0.46 km2, 1,160 m above sea level) is 100 ha of secondary rain forest and lies 23 km off Entebbe in the north‐west of Lake Victoria, and is part of the Koome group of islands, in the Mukono District, Uganda. By the time of sample collection, the group consisted of 23 females and 19 males with age group categorized as follows: 4 Infants (1–5years), 6 Juveniles (6–8 years), 12 sub‐adults (9– 11 years) and adults (12 years and more). They are managed semi‐intensively with supplementary feeding twice a day using domestically purchased fruits and vegetables. Specimen Collection Sterile oral swabs were collected from 39 chimpanzees of Ngamba Island Chimpanzee Sanctuary (NIC) during the annual health checks under anesthesia. None of the chimpanzee was ill at time of sample collection and 2 months prior to sample collection. The oral swabs were preserved in RNALater and stored at 80°C till the time of examination. Other nasopharyngeal swabs were taken from all chimpanzees and placed in Stewart’s transport medium immediately and transported to the Medical Microbiology Laboratory of the Medical School of Makerere University the same day within a few hours for culturing. The research study was approved by Uganda Wildlife Authority (UWA) and Uganda National Council of Science and Technology (UNCST). Non‐invasive samples were collected following the ethical and animal welfare standards according to Pan African Sanctuaries Alliance (PASA), International Primatological Society and American Society of Primatologists principals for ethical treatment of primates. The results of this study have been shared with sanctuaries through PASA Veterinary HealthCare workshops that then share information with respective governments. Bacterial Culturing Quality controlled media of Chocolate agar, 5% sheep blood agar and MacConkey agar with crystal violet plates were used in the study. These were inoculated with the nasopharyngeal samples and incubated at 35–37°C in 5% CO2 incubator except MacConkey which was incubated in an incubator at 35–37°C for 18–24 hr. On Chocolate agar plate a disk of Oleondomycin was placed to enable the growth of any Haemophilus spp. visible. After incubation the
Chimpanzee Nasopharyngeal Colonization / 105
plates were evaluated for growth and suspected colonies were subjected to identification. The gram‐ negative rods, after establishing a pure culture were identified using either API20E or API20NE (bioMerieux, France). Colonies of suspected S. pneumoniae were subjected to optochin test and bile solubility test.
fragments were purified by use of the QIAquick PCR purification kit (Qiagen). The purified PCR products from clones were sequenced and analyzed on an ABI Prism 3100 Genetic Analyzer [Sanger et al., 1977]. Sequence information obtained from the bacteria screenings were compared to sequences of the public databases for identification of the different species.
DNA Extraction
Phylogenetic Analysis
Nucleic acids DNA/RNA were extracted from oral swabs of 39 chimpanzees using QIAamp Viral RNA Min Kit (Qiagen®, QIAamp®, Hilden, Germany). Double extraction of DNA using the spin column protocol was used following manufactures instructions. The eluted DNA was stored in 1.5 Eppendorf tubes at 80°C till use for the next step.
Initial editing and alignment of amplified sequences were performed using SeqMan (www.dnastar.com). Edited sequences were analyzed using Ribosomal Database Project (RDP) analysis tools available online at http://rdp.cme.msu.edu [Cole et al., 2007; Maidak et al., 2001]. Thirty‐seven 16S rRNA gene sequences obtained from colony amplicons were first uploaded into SeqMatch version 3 that searches for its nearest neighbors and thereafter aligned and classified by myRDP analysis tool. 148,277 sequences were included in the search that is based on 7‐base oligomers. The data set was optimized for both type and non‐type strains, both environmental (uncultured) sequences and isolates, near full sequences (>1,200 bases) and of good quality sequences. Results from query sequences under bacteria were analyzed and those sequences classified to genus level recorded. The sequences were classified using RDP Classifier, a Naïve Bayesian Classifier for rapid assignment of rRNA sequences into the New Bacterial Taxonomy in Bergey’s Taxonomic Outline of Prokaryotes [Wang et al., 2007]. Each sequence was labeled with a set of taxa, from the domain to genus. The major taxonomic ranks are: domain, phylum, class, order, family, genus and species with occasional intermediate ranks such as “subclass” and “suborder.” SeqMatch, myRDP and/or public RDP sequences were used to create a phylogenetic tree using the Weighbor weighted neighbor‐joining tree building algorithm [Bruno et al., 2000; Cole et al., 2007]. The tree includes results from a bootstrap test using 100 replicates. Bootstraps higher than 50% are highlighted. The integrity of the amplified sequences displaying 99% identical. These include Haemophilus influenza, Neisseria menengitides, S. pnuemoniae, Kingella kingae, Nicotiana tabacum, Veillonella oral clone, and Provetella sp. Oral clone (Fig. 1). In addition, we analyzed the same samples using bacteriological culture methods and found that the oral cavity of the wild‐born captives chimpanzees in a sanctuary are colonized by both normal and potential
pathogenic bacteria with >50% harboring Klebsiella pneumoniae and Pseudomonas auruginosa (Table II). Further analysis using specific TaqMan PCR for S. pneumoniae performed on 38 chimpanzee oral samples revealed that 78.9% (30/38) chimpanzees were positive for S. pneumoniae. DISCUSSION Provision of proper care, restoration of health and socio‐psycho well‐being of newly rescued and traumatized orphaned apes is a priority management intervention in great ape sanctuaries. Frequent outbreaks of the respiratory related illnesses presents a big challenge to the management and care of ape communities especially newly arrived individuals. In this study, we analyzed the nasopharyngeal colonization of chimpanzees on Ngamba Island and found chimpanzee nasal pharynx colonized by four major phyla: Proteobacteria, Fermicutes, Bacteriodes, and Cynobacteria. Seventeen (45.9%) out of 37 amplified 16S rRNA gene clones could not be identified to genus level and were classified as unclassified Pasteurellaceae, Bacteriodales, and Lactobacillales. This group represents uncharacterized sequences 1,200 nucleotides in length within the RDP database. This is expected; as little or no work has been done on bacterial flora of chimpanzees and these sequences may be different bacterial species not cultivated and characterized previously. This data constitutes the nasopharyngeal colonization of chimpanzees previously unknown. Bacteriological cultivation method revealed more information bacterial species (gram positive and negative) colonizing the oral cavity of chimpanzees dominated by K. pneumonie (54.7%) and P. aeruginosa (50%; Table I) While TaqMan PCR, revealed that 78.9% (30/38) chimpanzee’s oral cavity to be
TABLE I. Bacterial Culture Results From 42 Wild‐Born Semi‐Captive Chimpanzees on Ngamba Island
Cultured bacteria
Positive chimpanzees
Gram stain
Pseudomonas aeruginosa
21 (50%)
Negative rods
Klebsiella pneumonie Bacillus Escherichia coli Staphylococcus aureus
23 12 17 3
Neg. coccobacilli Positive rods Negative rods Positive cocci
(54.7%) (28.5%) (40.5%) (7.1%)
Streptococcus pyogenes Proteus mirabilis
1(2.4%9 2 (4.7%)
Positive cocci Negative bacillus
Enterobacter aerogenes Other un‐identified bacteria
4 (9.5%)
Negative rods Both neg. cocci and rods
Am. J. Primatol.
Colony morphology description Large, green pyocanin pigment with irregular spreading margins and fruit like odour Mucoid, raised, thick, and watery Large, flat, white, serrated edge, and glistening Small, mucoid, entire, raised, Blue colonies on ECA, Round, glistening, smooth, opaque with golden pigmentation Round to ovoid cocci in chains/pairs Small rod‐like cells with characteristic concentric rings of growth Round smooth, convex white rods Pink colonies to mucoid on ECA
Chimpanzee Nasopharyngeal Colonization / 107
Fig. 1. Phylogenetic analysis of bacteria diversity detected in the oral cavity of healthy semi‐captive chimpanzees using 16S rRNA analysis. The color‐coded names of chimpanzees followed by accession number of bacteria spp. was used in branches. This tree was rooted by using an Actinomyces denticolens (T) 16S rRNA sequence as an out‐group (Accession no. NCTC 11490). The scale bar represents an expected nucleotide substitution rate of 1%.
colonized by S. pneumoniae. Interestingly, some of the identified bacterial species or phylotypes by all methods are known potential respiratory pathogens in humans (K. pneumoniae, S. pnuemoniae, H. influenza and Neisseria meningitides). The study shows that the nasopharynx of chimpanzees is colonized by both non‐pathogenic and pathogenic bacteria. The nasopharyngeal carriage of potential pathogenic bacteria in healthy semi‐captive chimpanzees is likely to be influenced by close contact with
caregivers and the diet that is composed of domestic fruits and vegetables purchased in local markets. This colonization presents risk factors to the development of respiratory diseases in chimpanzees especially in periods of stress. Our results are similar with documented human studies where human nasopharynx has been shown to be colonized by potential respiratory pathogens H. influenza, S. pnuemoniae and Moraxella catarrhalis in early childhood [Barbosa‐Cesnik et al., 2006; Darboe
Am. J. Primatol.
108 / Mugisha et al.
TABLE II. Classification of 28 Isolates Sequences From Oral Swabs of Four Chimpanzees Using Naïve Bayesian rRNA Classifier Version 1.0 No. 100% similarity of Classified bacterial isolates to genus level Chimp name
Streptococcus
Neisseria
Veillonella
Kingella
Asega
4
—
2
—
Okech
1
2
—
—
3 8 28.5
3 2 7 25.0
2 7.1
1 1 3.6
Kisembo Umutama Total isolates % of isolates
Unclassified bacteria group
