Rapid identification and differentiation of Mycobacterium avium subspecies paratuberculosis types by use of real-time PCR and high-resolution melt analysis of the MAP1506 locus

High-resolution melt (HRM) analysis can identify sequence polymorphisms by comparing the melting curves of amplicons generated by real-time PCR amplification. We describe the application of this technique to identify Mycobacterium avium subspecies paratuberculosis types I, II, and III. The HRM approach was based on type-specific nucleotide sequences in MAP1506, a member of the PPE (proline-proline-glutamic acid) gene family.

Occurrence of Mycobacterium avium subspecies paratuberculosis across host species and European countries with evidence for transmission between wildlife and domestic ruminants

BACKGROUND Mycobacterium avium subspecies paratuberculosis (Map) causes an infectious chronic enteritis (paratuberculosis or Johne's disease) principally of ruminants. The epidemiology of Map is poorly understood, particularly with respect to the role of wildlife reservoirs and the controversial issue of zoonotic potential (Crohn's disease). Genotypic discrimination of Map isolates is pivotal to descriptive epidemiology and resolving these issues. This study was undertaken to determine the genetic diversity of Map, enhance our understanding of the host range and distribution and assess the potential for interspecies transmission. RESULTS 164 Map isolates from seven European countries representing 19 different host species were genotyped by standardized IS900--restriction fragment length polymorphism (IS900-RFLP), pulsed-field gel electrophoresis (PFGE), amplified fragment length polymorphisms (AFLP) and mycobacterial interspersed repeat unit-variable number tandem repeat (MIRU-VNTR) analyses. Six PstI and 17 BstEII IS900-RFLP, 31 multiplex [SnaBI-SpeI] PFGE profiles and 23 MIRU-VNTR profiles were detected. AFLP gave insufficient discrimination of isolates for meaningful genetic analysis. Point estimates for Simpson's index of diversity calculated for the individual typing techniques were in the range of 0.636 to 0.664 but a combination of all three methods increased the discriminating power to 0.879, sufficient for investigating transmission dynamics. Two predominant strain types were detected across Europe with all three typing techniques. Evidence for interspecies transmission between wildlife and domestic ruminants on the same property was demonstrated in four cases, between wildlife species on the same property in two cases and between different species of domestic livestock on one property. CONCLUSION The results of this study showed that it is necessary to use multiple genotyping techniques targeting different sources of genetic variation to obtain the level of discrimination necessary to investigate transmission dynamics and trace the source of Map infections. Furthermore, the combination of genotyping techniques may depend on the geographical location of the population to be tested. Identical genotypes were obtained from Map isolated from different host species co-habiting on the same property strongly suggesting that interspecies transmission occurs. Interspecies transmission of Map between wildlife species and domestic livestock on the same property provides further evidence to support a role for wildlife reservoirs of infection.

Taxonomy and redescription of the Yellow-footed Antechinus, Antechinus flavipes (Waterhouse) (Marsupialia: Dasyuridae)

We provide a taxonomic redescription of the ubiquitous and variable dasyurid marsupial Yellow-footed Antechinus, Antechinus flavipes (Waterhouse), which comprises three currently recognized subspecies whose combined geographic distribution spans almost the length and breadth of Australia. A. flavipes leucogaster Gray is confined to south-west Western Australia; A. flavipes flavipes is distributed in south-eastern Australia across four states—South Australia, Victoria, New South Wales and Queensland; A. flavipes rubeculus Van Dyck is confined to the wet tropics of Queensland. A. flavipes is readily distinguished from all extant congeners based on external morphology by the following combination of features: a grey head; orange-yellow toned flanks/rump, feet and tail base; pale eye-rings and a darkened tail tip. A. flavipes skulls are stout, being broad at the level of the rear upper molars, have small palatal vacuities and small entoconid cusps on the lower molars. However, notable differences among subspecies of A. flavipesprevent any obvious collection of skull characters being diagnostic for species-level discrimination among congeners. A. flavipes rubeculus is the largest of the three subspecies of Yellow-footed Antechinus and most similar in skull morphology to A. leo, A. bellus and A. godmani—all four species are geographically limited to tropical Australia. A. f. rubeculus is notably larger in many characters than its conspecifics: A. f. flavipes, the next largest, and A. f. leucogaster, the smallest of the group. A. f. flavipes and A. f. leucogaster diverge significantly at only a few skull characters, and both subspecies have cranial morphological affinities with the recently discovered A. mysticus, most notably A. f. leucogaster. Phylogenies generated from mt- and nDNA data strongly support Antechinus flavipes as monophyletic with respect to other members of the genus; within A. flavipes, each of the three recognized subspecies form distinctive monophyletic clades.

Taxonomy and redescription of the Atherton Antechinus, Antechinus godmani (Thomas) (Marsupialia: Dasyuridae)

We provide a taxonomic redescription of the dasyurid marsupial Atherton Antechinus, Antechinus godmani (Thomas). A. godmani is only rarely encountered and limited to wet tropical rainforests of north-east Queensland, Australia, between the towns of Cardwell and Cairns (a distribution spanning 135 kilometres from north to south). The distinctive species occurs at altitudes of over 600 meters asl, in all major rainforest types, and can be found with both the northern subspecies of the Yellow-footed Antechinus, A. flavipes rubeculus Van Dyck and the Rusty Antechinus, A. adustus (Thomas). A. god-mani is clearly separated from all congeners on the basis of both morphometrics and genetics. A. godmani can be distin-guished from all extant congeners based on external morphology by a combination of large size, naked-looking tail and reddish fur on the face and head. A. godmani skulls are characteristically large, with a suite of long features: basicranium, palate, upper premolar tooth row, inter-palatal vacuity distance and dentary. Phylogenies generated from mt- and nDNA data position Antechinus godmani as monophyletic with respect to other members of the genus; A. godmani is strongly supported as the sister-group to a clade containing all other antechinus, but excluding the south-east Australian Dusky An-techinus, A. swainsonii (Waterhouse) and Swamp Antechinus, A. minimus (Geoffroy). Antechinus godmani are genetically very divergent compared to all congeners (mtDNA: range 12.9–16.3%).

The Black-tailed Antechinus, Antechinus arktos sp. nov.: a new species of carnivorous marsupial from montane regions of the Tweed Volcano caldera, eastern Australia

We describe a new species of dasyurid marsupial within the genus Antechinus that was previously known as a northern outlier of Dusky Antechinus (A. swainsonii). The Black-tailed Antechinus, Antechinus arktos sp. nov., is known only from areas of high altitude and high rainfall on the Tweed Volcano caldera of far south-east Queensland and north-east New South Wales, Australia. Antechinus arktos formerly sheltered under the taxonomic umbrella of A. swainsonii mimetes, the widespread mainland form of Dusky Antechinus. With the benefit of genetic hindsight, some striking morphological differences are herein resolved: A. s. mimetes is more uniformly deep brown-black to grizzled grey-brown from head to rump, with brownish (clove brown—raw umber) hair on the upper surface of the hindfoot and tail, whereas A. arktos is more vibrantly coloured, with a marked change from greyish-brown head to orange-brown rump, fuscous black on the upper surface of the hindfoot and dense, short fur on the evenly black tail. Further, A. arktos has marked orange-brown fur on the upper and lower eyelid, cheek and in front of the ear and very long guard hairs all over the body; these characters are more subtle in A. s. mimetes. There are striking genetic differences between the two species: at mtDNA, A. s. mimetes from north-east New South Wales is 10% divergent to A. arktos from its type locality at Springbrook NP, Queensland. In contrast, the Ebor A. s. mimetes clades closely with conspecifics from ACT and Victoria. A. arktos skulls are strikingly different to all subspecies of A. swainsonii. A. arktos are markedly larger than A. s. mimetes and A. s. swainsonii (Tasmania) for a range of craniodental measures. Antechinus arktos were historically found at a few proximate mountainous sites in south-east Queensland, and have only recently been recorded from or near the type locality. Even there, the species is likely in low abundance. The Black-tailed Antechinus has plausibly been detrimentally affected by climate change in recent decades, and will be at further risk with increasing warming trends.

Combined analysis of categorical and numerical descriptors of Australian groundnut accessions using nonlinear principal component analysis

For users of germplasm collections, the purpose of measuring characterization and evaluation descriptors, and subsequently using statistical methodology to summarize the data, is not only to interpret the relationships between the descriptors, but also to characterize the differences and similarities between accessions in relation to their phenotypic variability for each of the measured descriptors. The set of descriptors for the accessions of most germplasm collections consists of both numerical and categorical descriptors. This poses problems for a combined analysis of all descriptors because few statistical techniques deal with mixtures of measurement types. In this article, nonlinear principal component analysis was used to analyze the descriptors of the accessions in the Australian groundnut collection. It was demonstrated that the nonlinear variant of ordinary principal component analysis is an appropriate analytical tool because subspecies and botanical varieties could be identified on the basis of the analysis and characterized in terms of all descriptors. Moreover, outlying accessions could be easily spotted and their characteristics established. The statistical results and their interpretations provide users with a more efficient way to identify accessions of potential relevance for their plant improvement programs and encourage and improve the usefulness and utilization of germplasm collections.

The analysis of large scale data taken from the world groundnut (Arachis hypogaea L.) germplasm collection. II. Two-way data with mixed data types

As a sequel to a paper that dealt with the analysis of two-way quantitative data in large germplasm collections, this paper presents analytical methods appropriate for two-way data matrices consisting of mixed data types, namely, ordered multicategory and quantitative data types. While various pattern analysis techniques have been identified as suitable for analysis of the mixed data types which occur in germplasm collections, the clustering and ordination methods used often can not deal explicitly with the computational consequences of large data sets (i.e. greater than 5000 accessions) with incomplete information. However, it is shown that the ordination technique of principal component analysis and the mixture maximum likelihood method of clustering can be employed to achieve such analyses. Germplasm evaluation data for 11436 accessions of groundnut (Arachis hypogaea L.) from the International Research Institute of the Semi-Arid Tropics, Andhra Pradesh, India were examined. Data for nine quantitative descriptors measured in the post-rainy season and five ordered multicategory descriptors were used. Pattern analysis results generally indicated that the accessions could be distinguished into four regions along the continuum of growth habit (or plant erectness). Interpretation of accession membership in these regions was found to be consistent with taxonomic information, such as subspecies. Each growth habit region contained accessions from three of the most common groundnut botanical varieties. This implies that within each of the habit types there is the full range of expression for the other descriptors used in the analysis. Using these types of insights, the patterns of variability in germplasm collections can provide scientists with valuable information for their plant improvement programs.

Recent surveys of bats (Mammalia: Chiroptera) from China. I. Rhinolophidae and Hipposideridae

We conducted surveys of bats in China between 1999 and 2007, resulting in the identification of at least 62 species. In this paper we present data on 19 species, comprising 12 species from the family Rhinolophidae and seven from the Hipposideridae. Rhinolophids captured were Rhinolophus affinis, R. ferrumequinum, R. lepidus, R. luctus, R. macrotis, R. siamensis, R. marshalli, R. rex, R. pearsonii, R. pusillus, R. sinicus and R. stheno. Because of extensive morphological similarities we question the species distinctiveness of R. osgoodi (may be conspecific with R. lepidus), R. paradoxolophus (which may best be treated as a subspecies of R. rex), R. huananus (probably synonymous with R. siamensis), and we are skeptical as to whether R. sinicus is distinct from R. thomasi. Hipposiderids captured were Hipposideros armiger, H. cineraceus, H. larvatus, H. pomona, H. pratti, Aselliscus stoliczkanus and Coelops frithii. Of these species, two rhinolophids (Rhinolophus marshalli and R. stheno) and one hipposiderid (Hipposideros cineraceus) represent new species records for China. We present data on species' ranges, morphology and echolocation call frequencies, as well as some notes on ecology and conservation status. China hosts a considerable diversity of rhinolophid and hipposiderid bats, yet threats to their habitats and populations are substantial.

Echolocation calls, wing shape, diet and phylogenetic diagnosis of the endemic Chinese bat Myotis pequinius

We describe the echolocation calls, flight morphology and diet of the endemic Chinese bat Myotis pequinius Thomas, 1908. Orientation calls are broadband, and reach low terminal frequencies. Diet comprised 80% beetles by volume. Wing shape and call design suggest that the bats fly in cluttered habitats, and the possession of moderately long ears and the dietary composition imply they forage at least sometimes by gleaning. Myotis pequinius resembles a larger Oriental version of the western Palaearctic species M. nattereri. Phylogenetic analysis based on sequences of the cytochrome b gene of mitochondrial DNA (1,140 base pairs) from a range of Palaearctic Myotis species confirmed that M. pequinius is close to the nattereri group, and is a sister-species to the eastern Palaearctic M. bombinus. One bat sequenced from China could not be identified from available species descriptions. It was smaller than M. pequinius, and also differed from it in sequence divergence by 6.7%, suggesting the existence of additional, cryptic taxonomic diversity in this group. Our phylogenetic analysis also supports the recognition of M. schaubi as a species distinct from M. nattereri in Transcaucasia and south-western Asia. Myotis nattereri tschuliensis is more closely related to M. schaubi than to M. nattereri, and is best considered either as a subspecies of M. schaubi, or possibly as a distinct species.

Phylogenetics of small horseshoe bats from east Asia based on mitochondrial DNA sequence variation

We undertook analyses of mitochondrial DNA gene sequences and echolocation calls to resolve phylogenetic relationships among the related bat taxa Rhinolophus pusillus (sampled across China), R. monoceros (Taiwan), R. cornutus (main islands of Japan), and R. c. pumilus (Okinawa, Japan), Phylogenetic trees and genetic divergence analyses were constructed by combining new complete mitochondrial cytochrome-b gene sequences and partial mitochondrial control region sequences with published sequences. Our work showed that these 4 taxa formed monophyletic groups in the phylogenetic tree. However, low levels of sequence divergence among the taxa, together with similarities in body size and overlapping echolocation call frequencies, point to a lack of taxonomic distinctiveness. We therefore suggest that these taxa are better considered as geographical subspecies rather than distinct species, although this should not diminish the conservation importance of these island populations, which are important evolutionarily significant units. Based on our findings, we suggest that the similarities in body size and echolocation call frequency in these rhinolophids result from their recent common ancestry, whereas similarities in body size and call frequency with R. hipposideros of Europe are the result of convergent evolution.

Search-phase echolocation calls of the New Zealand lesser short-tailed bat (Mystacina tuberculata) and long-tailed bat (Chalinolobus tuberculatus).

This paper describes the search-phase echolocation calls of lesser short-tailed bats (Mystacina tuberculata) and long-tailed bats (Chalinolobus tuberculatus). Calls were recorded from all three subspecies of short-tailed bat and seven populations of long-tailed bat, three in Northland, two in the central North Island, and two in the lower South Island. The calls were recorded in the field and digitised, then three spectral components and one temporal component of the calls were measured. Calls of the lesser short-tailed bat could be loosely classified into subspecies by means of multivariate discriminant function analysis. Similarly, long-tailed bat calls showed regional variation, and discriminant function analysis was able to fit calls to regional groups with a high rate of success. The significance of the results presented is discussed in terms of the conservation of New Zealand bats and the unique ecology of the lesser short-tailed bat.

A taxonomic assessment of the Australian Dusky Antechinus Complex: A new species, the Tasman Peninsula Dusky Antechinus (Antechinus vandycki sp. nov.) and an elevation to species of the Mainland Dusky Antechinus (Antechinus swainsonii mimetes (Thomas))

In 2014, the northern outlying population of carnivorous marsupial Dusky Antechinus (Antechinus swainsonii) was nominated a new species, A. arktos. Here, we describe a further new species in the dasyurid A. swainsonii complex, which now contains five taxa. We recognise two distinct species from Tasmania, formerly represented by A. swainsonii swainsonii (Waterhouse); one species (and 2 subspecies) from mainland south-eastern Australia, formerly known as A. swainsonii mimetes (Thomas) and A. swainsonii insulanus Davison; and one species from the Tweed Caldera in mid-eastern Australia, formerly known as A. s. mimetes but recently described as A. arktos Baker, Mutton, Hines and Van Dyck. Primacy of discovery dictates the Tasmanian Dusky Antechinus A. swainsonii (Waterhouse) is nominate; the Mainland Dusky Antechinus taxa, one raised from subspecies within A. swainsonii mimetes (Thomas) is elevated to species (now A. mimetes mimetes) and the other, A. swainsonii insulanus Davison is transferred as a subspecies of A. mimetes (now A. mimetes insulanus); a species from Tasmania, the Tasman Peninsula Dusky Antechinus, is named A. vandycki sp. nov. These taxa are strongly differentiated: geographically (in allopatry), morphologically (in coat colour and craniodental features) and genetically (in mtDNA, 7.5-12.5% between species pairs).

Ecology of uncultivated oscillospira species in the rumen of cattle, sheep, and reindeer as assessed by microscopy and molecular approaches

The ecology of the uncultured, but large and morphologically conspicuous, rumen bacterium Oscillospira spp. was studied. Oscillospira-specific 16S rRNA gene sequences were detected in North American domestic cattle, sheep from Australia and Japan, and Norwegian reindeer. Phylogenetic analysis of the sequences obtained allowed definition of three operational taxonomic units within the Oscillospira clade. Consistent with this genetic diversity, we observed atypical smaller morphotypes by using an Oscillospira-specific fluorescence in situ hybridization probe. Despite the visual disappearance of typical large Oscillospira morphotypes, the presence of Oscillospira spp. was still detected by Oscillospira-specific PCR in the rumen of cattle and sheep. These observations suggest the broad presence of Oscillospira species in various rumen ecosystems with the level, and most likely the morphological form, dependent on diet. An ecological analysis based on enumeration of the morphologically conspicuous, large-septate form confirms that the highest counts are associated with the feeding of fresh forage diets to cattle and sheep and in two different subspecies of reindeer investigated.

Cuticular Hydrocarbons of Buffalo Fly, Haematobia exigua, and Chemotaxonomic Differentiation from Horn Fly, H.irritans

We determined the quantity and chemical composition of cuticular hydrocarbons of different strains, sex and age of buffalo flies, Haematobia exigua. The quantity of cuticular hydrocarbons increased from less than 1 µg/fly for newly-emerged flies to over 11 µg/fly in 13 d-old flies. The hydrocarbon chain length varied from C21 to C29, with unbranched alkanes and monounsaturated alkenes the major components. Newly emerged flies produced almost exclusively C27 hydrocarbons. Increasing age was accompanied by the appearance of hydrocarbons with shorter carbon chains and an increase in the proportion of alkenes. 11 Tricosene and 7-tricosene were the most abundant hydrocarbons in mature buffalo flies. Cuticular hydrocarbons of buffalo flies are distinctly different from those of horn flies. The most noticeable differences were in the C23 alkenes, with the major isomers 11- and 7-tricosene in buffalo flies and (Z)-9- and (Z)-5-tricosene in horn flies, respectively. Cuticular hydrocarbon analysis provides a reliable method to differentiate buffalo and horn fly, which are difficult to separate morphologically. The differences in cuticular hydrocarbons also support their recognition as separate species, H. exigua and H. irritans, rather than as subspecies.