Biomedical subjects

Robert E Mandrell

Publications and source records attributed to Robert E Mandrell.

At least 19 recordsLinked to original sources

PubMed2006

The absence of intervening sequences in 23S rRNA genes of Campylobacter coli isolates from Turkeys is a unique attribute of a cluster of related strains which also lack resistance to erythromycin.

Certain Campylobacter strains harbor a transcribed intervening sequence (IVS) in their 23S rRNA genes. Following transcription, the IVS is excised, leading to fragmentation of the 23S rRNA. The origin and possible functions of the IVS are unknown. Furthermore, the distribution of IVS-harboring strains within Campylobacter populations is poorly understood. In this study, 104 strains of Campylobacter coli from turkeys, representing 27 different multilocus sequence typing-based sequence types (STs), were characterized in terms of IVS content and erythromycin susceptibility. Sixty-nine strains harbored IVSs in all three 23S rRNA genes, whereas the other 35 strains lacked IVSs from at least one of the genes. The STs of the latter strains belonged to an unusual cluster of C. coli STs (cluster II), earlier found primarily in turkey strains and characterized by the presence of the C. jejuni aspA103 allele. The majority (66/69) of strains harboring IVSs in all three 23S rRNA genes were resistant to erythromycin, whereas none of the 35 strains with at least one IVS-free 23S rRNA gene were resistant. Cluster II strains could be transformed to erythromycin resistance with genomic DNA from C. coli that harbored IVS and the A2075G transition in the 23S rRNA gene, associated with resistance to erythromycin in Campylobacter. Erythromycin-resistant transformants harbored both the A2075 transition and IVS. The findings suggest that the absence of IVS in C. coli from turkeys is characteristic of a unique clonal group of erythromycin-susceptible strains and that IVS can be acquired by these strains via natural transformation to erythromycin resistance.

Animals↗

PubMed2006

Cryptic plasmids isolated from Campylobacter strains represent multiple, novel incompatibility groups.

Three small, cryptic plasmids from the multi-drug-resistant (MDR) Campylobacter coli strain RM2228 and one small, cryptic plasmid from the MDR Campylobacter jejuni strain RM1170 were sequenced and characterized. pCC2228-1 has some similarity to Firmicutes RepL family plasmids that replicate via a rolling-circle mechanism. pCC2228-2 is a theta-replicating, iteron-containing plasmid (ICP) that is a member of the same incompatibility (Inc) group as previously described Campylobacter shuttle vectors. The other two ICPs, pCC2228-3 and pCJ1170, represent a second novel Inc group. Comparison of the four plasmids described in this study with other characterized plasmids from C. jejuni, C. coli, C. lari, and C. hyointestinalis suggests that cryptic plasmids in Campylobacter may be classified into as many as nine Inc groups. The plasmids characterized in this study have several unique features suitable for the construction of novel Campylobacter shuttle vectors, e.g., small size, absence of many common multiple-cloning site restriction sites, and Inc groups not represented by current Campylobacter shuttle plasmids. Thus, these plasmids may be used to construct a new generation of Campylobacter shuttle vectors that would permit transformation of environmental Campylobacter isolates with an existing repertoire of native plasmids.

Bacterial Proteins↗

PubMed2006

Listeria monocytogenes subgroups IIIA, IIIB, and IIIC delineate genetically distinct populations with varied pathogenic potential.

Listeria monocytogenes lineage III strains belonging to subgroups IIIA (n = 8), IIIB (n = 5), and IIIC (n = 6) were examined along with other known serotype strains (n = 11) by PCR and Southern hybridization using several recently described species-, virulence-, and serotype-specific primers and probes. The virulence of seven representative lineage III strains was then evaluated in mice via the intraperitoneal route. The results suggest that subgroup IIIA consists of typical rhamnose-positive avirulent serotype 4a and virulent serotype 4c strains, subgroup IIIC consists of atypical rhamnose-negative virulent serotype 4c strains, and subgroup IIIB consists of atypical rhamnose-negative virulent non-serotype 4a and non-serotype 4c strains, some of which may be related to serotype 7. It is possible that subgroup IIIB (including serotype 7) may represent a novel subspecies within L. monocytogenes.

Animals↗

PubMed2006

Comparative genomic analysis of Campylobacter jejuni strains reveals diversity due to genomic elements similar to those present in C. jejuni strain RM1221.

Analysis of the complete genomic sequence of Campylobacter jejuni strain RM1221 identified four large genomic elements, Campylobacter jejuni-integrated elements (CJIEs), that were absent from C. jejuni strain NCTC 11168. To further investigate the genomic diversity of Campylobacter, we conducted a comparative genomic analysis from a collection of 67 C. jejuni and 12 Campylobacter coli strains isolated from various geographical locations and clinical and veterinary sources. Utilizing PCR, we demonstrated that 55% of the C. jejuni strains examined were positive for at least one RM1221-like genomic element and 27% were positive for two or more of these CJIEs. Furthermore, many C. coli strains were positive for either genomic element CJIE1 or CJIE3. To simultaneously assess for the presence or absence of several genes that comprise the various CJIEs, we developed a multistrain C. jejuni DNA microarray that contained most of the putative coding sequences for strains NCTC 11168 and RM1221. A comparative genomic hybridization (CGH) analysis of 35 of the 67 C. jejuni strains confirmed the presence of genomic elements similar to those in strain RM1221. Interestingly, the DNA microarray analysis demonstrated that these genomic elements in the other C. jejuni strains often exhibited modular patterns with some regions of the CJIEs present and other regions either absent or highly divergent compared to strain RM1221. Our CGH method also identified 18 other intraspecies hypervariable regions, such as the capsule and lipooligosaccharide biosynthesis regions. Thus, the inclusion of genes from these integrated genomic elements and the genes from the other intraspecies hypervariable regions contributes to a better assessment of the diversity in C. jejuni and may increase the usefulness of DNA microarrays as an epidemiological genotyping tool. Finally, we also showed that in CJIE1, a Campylobacter Mu-like phage, is located differentially in other strains of C. jejuni, suggesting that it may integrate essentially randomly.

Campylobacter jejuni↗

PubMed2006

Identification of host-associated alleles by multilocus sequence typing of Campylobacter coli strains from food animals.

Campylobacter coli is a food-borne pathogen associated increasingly with human gastroenteritis. C. coli has a high prevalence in swine, but is isolated also from cattle and poultry. Multilocus sequence typing (MLST) systems have been developed to differentiate C. coli strains. Although substantial allelic diversity was identified across all seven C. coli MLST loci, no correlations were made in two previous studies between allele or sequence type (ST) and the source of the organism. However, this may be due to either the relatively small number or the low diversity of C. coli strains used to validate both MLST studies. This study describes the typing of 488 C. coli strains from 4 different food animal sources (cattle, chickens, swine and turkeys), collected at different times over a 6 year period from different USA geographical locations. A total of 149 STs were identified. The 185 swine strains were the most diverse, possessing 82 STs. The cattle strains were the most clonal; 52/63 (83 %) strains possessed a single ST (ST-1068). A subpopulation of C. coli strains, collected primarily from turkeys, was identified, containing both C. coli- and Campylobacter jejuni-associated MLST alleles, specifically the C. jejuni allele aspA103. The majority of STs and alleles were host associated, i.e. found primarily in strains from a single food-animal source. Only 12/149 (8 %) STs were found in multiple sources. Additionally, the majority (34/46, 74 %) of major (n>5) alleles were more prevalent in certain hosts (swine, poultry). The presence of host-associated C. coli MLST alleles could lead potentially to more efficient source tracking in this species, especially in the trace-back of both sporadic and outbreak human clinical C. coli strains to animal sources.

Alleles↗

PubMed2006

Competitive fitness of Listeria monocytogenes serotype 1/2a and 4b strains in mixed cultures with and without food in the U.S. Food and Drug Administration enrichment protocol.

Thirteen different serotypes of the food-borne pathogen Listeria monocytogenes have been described. Serotype 4b strains are most often associated with illness, and serotype 1/2a strains are most often isolated from foods and processing plants. Different abilities to respond to stresses have been described for serotype 4b and 1/2a strains. One of the common enrichment protocols used to test foods for the presence L. monocytogenes is described in the U.S. Food and Drug Administration (FDA) Bacterial Analytical Manual (BAM). We compared three strains of L. monocytogenes serotype 4b and five strains of serotype 1/2a in direct competition with each other in two-strain mixed cultures by using the FDA BAM enrichment protocol, which includes both enrichment broth and selective agar, with and without added food to mimic the conditions that occur during attempts to isolate Listeria species from contaminated foods. Using a colony immunoblot procedure and analyzing over 112,000 colonies, we observed differences in strain fitness, but these differences were not attributable to serotype or genetic lineage.

Animals↗

PubMed2006

Listeria monocytogenes serotype 4b strains belonging to lineages I and III possess distinct molecular features.

A collection of Listeria monocytogenes serotype 4b strains belonging to lineages I and III were examined by PCR and Southern blot analysis using species-, virulence-, and serotype-specific primers and probes. Whereas four serotype 4b lineage I strains reacted in PCR with the serotype 4b-, 4d-, and 4e-specific ORF2110 and virulence-specific lmo1134 and lmo2821 primers, all nine serotype 4b lineage III strains were negative by ORF2110 and lmo1134 primers. In addition, the nine serotype 4b lineage III strains formed two separate groups through their reactions in PCR with virulence-specific lmo2821 primers. Southern blot analysis using species-specific lmo0733 and virulence-specific lmo2821 gene probes largely confirmed the PCR results. These findings indicate that L. monocytogenes serotype 4b strains belonging to lineages I and III possess distinct molecular features.

Animals↗

PubMed2006

Escherichia coli O157:H7 survival and growth on lettuce is altered by the presence of epiphytic bacteria.

Escherichia coli O157:H7 can survive in low numbers in soil and on plants. Occasionally, conditions may occur in the field that lead to contamination of produce. Survival of enteric pathogens in the field is controlled to a certain extent by complex interactions with indigenous soilborne and seedborne epiphytes. Identifying these interactions may assist in developing strategies to improve produce safety. Two epiphytes were isolated from pathogen-contaminated plants that interact differently with E. coli O157:H7. Wausteria paucula enhanced the survival of E. coli O157:H7 six-fold on lettuce foliage grown from coinoculated lettuce seed. In contrast, Enterobacter asburiae decreased E. coli O157:H7 survival 20- to 30-fold on foliage. Competition also occurred in the rhizosphere and in plant exudate. This competition may be the result of E. asburiae utilization of several of the carbon and nitrogen substrates typically present in exudate and also used by E. coli O157:H7. Hence, competition observed on the plant may involve one or more nutrients provided by the plant. In contrast, a different mechanism may exist between E. coli O157:H7 and W. paucula since commensalism was only observed on foliage, not in the rhizosphere or plant exudate. Good agricultural practices that encourage the growth of competing bacteria, like E. asburiae, may reduce the incidence of produce contamination.

Antibiosis↗

PubMed2006

Antimicrobial activities of tea catechins and theaflavins and tea extracts against Bacillus cereus.

We evaluated the antimicrobial activities of seven green tea catechins and four black tea theaflavins, generally referred to as flavonoids, as well as the aqueous extracts (infusions) of 36 commercial black, green, oolong, white, and herbal teas against Bacillus cereus (strain RM3190) incubated at 21 degrees C for 3, 15, 30, and 60 min. The results obtained demonstrate that (i) (-)-gallocatechin-3-gallate, (-)-epigallocatechin-3-gallate, (-)-catechin-3-gallate, (-)-epicatechin-3-gallate, theaflavin-3, 3'-digallate, theaflavin-3'-gallate, and theaflavin-3-gallate showed antimicrobial activities at nanomolar levels; (ii) most compounds were more active than were medicinal antibiotics, such as tetracycline or vancomycin, at comparable concentrations; (iii) the bactericidal activities of the teas could be accounted for by the levels of catechins and theaflavins as determined by high-pressure liquid chromatography; (iv) freshly prepared tea infusions were more active than day-old teas; and (v) tea catechins without gallate side chains, gallic acid and the alkaloids caffeine and theobromine also present in teas, and herbal (chamomile and peppermint) teas that contain no flavonoids are all inactive. These studies extend our knowledge about the antimicrobial effects of food ingredients.

Anti-Bacterial Agents↗

PubMed2005

Genomic and proteomic identification of a DNA-binding protein used in the "fingerprinting" of campylobacter species and strains by MALDI-TOF-MS protein biomarker analysis.

We have identified a prominent approximately 10-kDa protein biomarker observed in the matrix-assisted laser desorption/ionization time-of-flight mass spectra (MALDI-TOF-MS) of cell lysates of five thermophilic species of Campylobacter: jejuni, coli, lari, upsaliensis, and helveticus. The biomarker was unambiguously identified by genomic and proteomic sequencing as a DNA-binding protein HU. We report the amino acid sequence of HU as determined by sequencing the hup gene of four species (12 strains): C. jejuni (2), C. coli (4), C. upsaliensis (4) and C. lari(2). Confirmation of the amino acid sequence was obtained by nanoflow high-performance liquid chromatography-tandem mass spectrometry of the tryptic peptides of the extracted/digested HU protein. Protein identification was also confirmed by comparison of the molecular weight (MW) predicted from the hup gene and the MW of HU as measured by high-resolution mass spectrometry. We found the HU protein to be particularly useful as a biomarker in that it strongly ionizes by MALDI and its MW varies between species and among strains within a species. Intra- and interspecies variation of the HU MW is due to changes in the amino acid sequence of the HU protein and not due to co- or posttranslational modifications. The strong ionization efficiency of HU by MALDI is likely due, in part, to four lysine residues clustered at the carboxyl end of the protein. We also report identification of the HU protein biomarker for a C. helveticus strain, whose hup gene was not sequenced, but whose HU amino acid sequence was partially conserved in C. upsaliensis strains. We have also tentatively assigned a approximately 10.5-kDa protein biomarker of a C. concisus strain as an HU protein.

Amino Acid Sequence↗

PubMed2005

Major structural differences and novel potential virulence mechanisms from the genomes of multiple campylobacter species.

Sequencing and comparative genome analysis of four strains of Campylobacter including C. lari RM2100, C. upsaliensis RM3195, and C. coli RM2228 has revealed major structural differences that are associated with the insertion of phage- and plasmid-like genomic islands, as well as major variations in the lipooligosaccharide complex. Poly G tracts are longer, are greater in number, and show greater variability in C. upsaliensis than in the other species. Many genes involved in host colonization, including racR/S, cadF, cdt, ciaB, and flagellin genes, are conserved across the species, but variations that appear to be species specific are evident for a lipooligosaccharide locus, a capsular (extracellular) polysaccharide locus, and a novel Campylobacter putative licABCD virulence locus. The strains also vary in their metabolic profiles, as well as their resistance profiles to a range of antibiotics. It is evident that the newly identified hypothetical and conserved hypothetical proteins, as well as uncharacterized two-component regulatory systems and membrane proteins, may hold additional significant information on the major differences in virulence among the species, as well as the specificity of the strains for particular hosts.

Animals↗

PubMed2005

Diversity within the Campylobacter jejuni type I restriction-modification loci.

The type I restriction-modification (hsd) systems of 73 Campylobacter jejuni strains were characterized according to their DNA and amino acid sequences, and/or gene organization. A number of new genes were identified which are not present in the sequenced strain NCTC 11168. The closely related organism Helicobacter pylori has three type I systems; however, no evidence was found that C. jejuni strains contain multiple type I systems, although hsd loci are present in at least two different chromosomal locations. Also, unlike H. pylori, intervening ORFs are present, in some strains, between hsdR and hsdS and between hsdS and hsdM. No definitive function can be ascribed to these ORFs, designated here as rloA-H (R-linked ORF) and mloA-B (M-linked ORF). Based on parsimony analysis of amino acid sequences to assess character relatedness, the C. jejuni type I R-M systems are assigned to one of three families: 'IAB', 'IC' or 'IF'. This study confirms that HsdM proteins within a family are highly conserved but share little homology with HsdM proteins from other families. The 'IC' hsd loci are >99 % identical at the nucleotide level, as are the 'IF' hsd loci. Additionally, whereas the nucleotide sequences of the 'IAB' hsdR and hsdM genes show a high degree of similarity, the nucleotide sequences of the 'IAB' hsdS and rlo genes vary considerably. This diversity suggests that recombination between 'IAB' hsd loci would lead not only to new hsdS alleles but also to the exchange of rlo genes; five C. jejuni hsd loci are presumably the result of such recombination. The importance of these findings with regard to the evolution of C. jejuni type I R-M systems is discussed.

Animals↗

PubMed2005

Speciation of Campylobacter coli, C. jejuni, C. helveticus, C. lari, C. sputorum, and C. upsaliensis by matrix-assisted laser desorption ionization-time of flight mass spectrometry.

Multiple strains of Campylobacter coli, C. jejuni, C. helveticus, C. lari, C. sputorum, and C. upsaliensis isolated from animal, clinical, or food samples have been analyzed by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). Whole bacterial cells were harvested from colonies or confluent growth on agar and transferred directly into solvent and then to a spot of dried 3-methoxy-4-hydroxycinnamic acid (matrix). Multiple ions in the 5,000- to 15,000-Da mass range were evident in spectra for each strain; one or two ions in the 9,500- to 11,000-Da range were consistently high intensity. "Species-identifying" biomarker ions (SIBIs) were evident from analyses of multiple reference strains for each of the six species, including the genome strains C. jejuni NCTC 11168 and C. jejuni RM1221. Strains grown on nine different combinations of media and atmospheres yielded SIBI masses within +/-5 Da with external instrument calibration. The highest-intensity C. jejuni SIBIs were cytosolic proteins, including GroES, HU/HCj, and RplL. Multiple intraspecies SIBIs, corresponding probably to nonsynonymous nucleotide polymorphisms, also provided some intraspecies strain differentiation. MALDI-TOF MS analysis of 75 additional Campylobacter strains isolated from humans, poultry, swine, dogs, and cats revealed (i) associations of SIBI type with source, (ii) strains previously speciated incorrectly, and (iii) "strains" composed of more than one species. MALDI-TOF MS provides an accurate, sensitive, and rapid method for identification of multiple Campylobacter species relevant to public health and food safety.

Animals↗

PubMed2005

Extended multilocus sequence typing system for Campylobacter coli, C. lari, C. upsaliensis, and C. helveticus.

A multilocus sequence typing (MLST) system has been reported previously for Campylobacter jejuni to both differentiate strains and identify clonal lineages. However, sequence variation at the MLST loci prevents its use for closely related Campylobacter species. We describe herein an expanded MLST method to include three clinically relevant Campylobacter species, C. coli, C. lari, and C. upsaliensis, and a fourth Campylobacter species, C. helveticus. The C. coli and C. helveticus methods use the same seven C. jejuni loci (aspA, atpA, glnA, gltA, glyA, pgm, and tkt); however, adk and pgi were substituted for aspA and gltA in C. lari and for gltA and pgm in C. upsaliensis. Multiple C. coli (n = 57), C. lari (n = 20), C. upsaliensis (n = 78), and C. helveticus (n = 9) isolates, representing both clinical and environmental sources, were typed. All four species were genetically diverse: the majority (> 80%) of the isolates had unique sequence types (STs). Using this method, mixed C. lari, C. upsaliensis, and C. helveticus isolates were identified; upon separation, each isolate was shown to contain two strains of the same species with distinct STs. Additionally, the expanded MLST method was able to detect potential lateral transfer events between C. jejuni and either C. coli or C. lari and between C. upsaliensis and C. helveticus. Thus, the expanded MLST method will prove useful in differentiating strains of five Campylobacter species, identifying mixed Campylobacter cultures, and detecting genetic exchange within the genus.

Base Sequence↗

PubMed2005

Comparison of Campylobacter jejuni lipooligosaccharide biosynthesis loci from a variety of sources.

Campylobacter jejuni strains exhibit significant variation in the genetic content of the lipooligosaccharide (LOS) biosynthesis loci with concomitant differences in LOS structure. The C. jejuni LOS loci have been grouped into six classes based on gene content and organization. Utilizing PCR amplifications of genes from these loci, we were able to classify a majority (80%) of the LOS biosynthesis loci from 123 strains of C. jejuni that included 39 of the Penner serotype reference strains. We found that a particular LOS class was not always associated with a specific Penner serotype, and 14 of 16 Guillain-Barré syndrome-associated isolates tested in this study shared the same LOS class. The remaining isolates that could not be classified were often distinguishable from each other based on the results of gene-specific PCR and the lengths of their LOS biosynthesis loci as determined by long (XL) PCR. Sequence analysis of two of these unique XL PCR products demonstrated two new LOS classes. These results support the hypothesis that the LOS locus is a hot spot for genetic exchange and rearrangements. Analysis of the LOS biosynthesis genes by PCR assays can be used for typing C. jejuni and offers the advantage of inferring potential LOS structures.

Animals↗

PubMed2004

Antibacterial activities of plant essential oils and their components against Escherichia coli O157:H7 and Salmonella enterica in apple juice.

We evaluated 17 plant essential oils and nine oil compounds for antibacterial activity against the foodborne pathogens Escherichia coli O157:H7 and Salmonella enterica in apple juices in a bactericidal assay in terms of % of the sample that resulted in a 50% decrease in the number of bacteria (BA(50)). The 10 compounds most active against E. coli (60 min BA(50) range in clear juice, 0.018-0.093%) were carvacrol, oregano oil, geraniol, eugenol, cinnamon leaf oil, citral, clove bud oil, lemongrass oil, cinnamon bark oil, and lemon oil. The corresponding compounds against S. enterica (BA(50) range, 0.0044-0.011%) were Melissa oil, carvacrol, oregano oil, terpeineol, geraniol, lemon oil, citral, lemongrass oil, cinnamon leaf oil, and linalool. The activity (i) was greater for S. enterica than for E. coli, (ii) increased with incubation temperature and storage time, and (iii) was not affected by the acidity of the juices. The antibacterial agents could be divided into two classes: fast-acting and slow-acting. High-performance liquid chromatography analysis showed that the bactericidal results are related to the composition of the oils. These studies provide information about new ways to protect apple juice and other foods against human pathogens.

Acrolein↗

PubMed2004

Comparison of survival of Campylobacter jejuni in the phyllosphere with that in the rhizosphere of spinach and radish plants.

Campylobacter jejuni has been isolated previously from market produce and has caused gastroenteritis outbreaks linked to produce. We have tested the ability of this human pathogen to utilize organic compounds that are present in leaf and root exudates and to survive in the plant environment under various conditions. Carbon utilization profiles revealed that C. jejuni can utilize many organic acids and amino acids available on leaves and roots. Despite the presence of suitable substrates in the phyllosphere and the rhizosphere, C. jejuni was unable to grow on lettuce and spinach leaves and on spinach and radish roots of plants incubated at 33 degrees C, a temperature that is conducive to its growth in vitro. However, C. jejuni was cultured from radish roots and from the spinach rhizosphere for at least 23 and 28 days, respectively, at 10 degrees C. This enteric pathogen also persisted in the rhizosphere of spinach for prolonged periods of time at 16 degrees C, a temperature at which many cool-season crops are grown. The decline rate constants of C. jejuni populations in the spinach and radish rhizosphere were 10- and 6-fold lower, respectively, than on healthy spinach leaves at 10 degrees C. The enhanced survival of C. jejuni in soil and in the rhizosphere may be a significant factor in its contamination cycle in the environment and may be associated with the sporadic C. jejuni incidence and campylobacteriosis outbreaks linked to produce.

Animals↗

PubMed2004

Intraspecific phylogeny and lineage group identification based on the prfA virulence gene cluster of Listeria monocytogenes.

Listeria monocytogenes is a serious food-borne pathogen that can cause invasive disease in humans and other animals and has been the leading cause of food recalls due to microbiological concerns in recent years. In order to test hypotheses regarding L. monocytogenes lineage composition, evolution, ecology, and taxonomy, a robust intraspecific phylogeny was developed based on prfA virulence gene cluster sequences from 113 L. monocytogenes isolates. The results of the multigene phylogenetic analyses confirm that L. monocytogenes comprises at least three evolutionary lineages, demonstrate that lineages most frequently (lineage 1) and least frequently (lineage 3) associated with human listeriosis are sister-groups, and reveal for the first time that the human epidemic associated serotype 4b is prevalent among strains from lineage 1 and lineage 3. In addition, a PCR-based test for lineage identification was developed and used in a survey of food products demonstrating that the low frequency of association between lineage 3 isolates and human listeriosis cases likely reflects rarity of exposure and not reduced virulence for humans as has been previously suggested. However, prevalence data do suggest lineage 3 isolates may be better adapted to the animal production environment than the food-processing environment. Finally, analyses of haplotype diversity indicate that lineage 1 has experienced a purge of genetic variation that was not observed in the other lineages, suggesting that the three L. monocytogenes lineages may represent distinct species within the framework of the cohesion species concept.

Animals↗