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Possible chinks in the crocodile armour: defining skin microflora
Abstract and Figures
Background
The global crocodilian skin market is currently in oversupply. As a result, the tanneries can now be very selective about the quality of skins they purchase. The challenge to producers is to meet these quality standards. A part of this challenge is to understand the risk of pathogens to crocodile skin quality.
Aims/objectives
Collectively, the aims of this project were to understand crocodile skin microflora and to better understand the threats to skin quality.
Methods used
To characterise skin microflora, 16SrRNA tag sequencing was used to compare wild and captive skins, whilst the identification of blemish causation was achieved using specific genetic sequences to screen for particular organisms. A survey of crocodile skins allowed the infection dynamics of poxvirus to be understood.
Results/key findings
Dermatophilus sp infection is not isolated to focal lesions as first thought. Dermatophilus sp. is also implicated, at high reads, in linear lesions. It continues to be problematic in focal lesions but can be more subtle than when first described as “brown spot”. Dermatophilus sp can be easily confused with early active poxvirus lesions. Wild saltwater crocodile have a substantially lower presence of Dermatophilus sp. suggesting that intensification has allowed it to develop an ecological niche in farm production settings.
While Dermatophilus sp. and poxvirus were identified from skin lesions with high prevalence, the Crocodyline herpesvirus and the Kunjin strain of West Nile virus were also identified. The prevalence of poxvirus is particularly high in grower pens but the infection dynamics on the two farms studied are contrary to each other. On one farm, poxvirus prevalence decreases as the animals approach finishing size and therefore has relatively little impact on production time. However, on the other farm, the number of lesions, particularly early stage lesions, increases and delays harvest times thus increasing production costs.
Implications and Recommendations
Developing quantitative genetic methods to detect these pathogens and assess different hygiene regimes is essential. This will allow producers to quantitatively assess the efficacy of their current regimes and make appropriate decisions. Reducing the impact of these pathogens will be crucial to delivering the skin quality demanded.
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Crocodilepox virus is a large dsDNA virus belonging to the genus Crocodylidpoxvirus, which infects a wide range of host species in the order Crocodylia worldwide. Here, we present genome sequences for a novel saltwater crocodilepox virus, with two subtypes (SwCRV-1 and -2), isolated from the Australian saltwater crocodile. Affected belly skins of juvenile saltwater crocodiles were used to sequence complete viral genomes, and perform electron microscopic analysis that visualized immature and mature virions. Analysis of the SwCRV genomes showed a high degree of sequence similarity to CRV (84.53% and 83.70%, respectively), with the novel SwCRV-1 and -2 complete genome sequences missing 5 and 6 genes respectively when compared to CRV, but containing 45 and 44 predicted unique genes. Similar to CRV, SwCRV also lacks the genes involved in virulence and host range, however, considering the presence of numerous hypothetical and or unique genes in the SwCRV genomes, it is completely reasonable that the genes encoding these functions are present but not recognized. Phylogenetic analysis suggested a monophyletic relationship between SwCRV and CRV, however, SwCRV is quite distinct from other chordopoxvirus genomes. These are the first SwCRV complete genome sequences isolated from saltwater crocodile skin lesions.
Reptilian skin is tough and scaled representing an evolutionary adaptation to the terrestrial environment. The presence of sulfhydryl oxidase during the process of hardening of the corneous layer in reptilian epidermis has been analyzed by immunocytochemistry and immunoblotting. Sulfhydryl oxidase-like immunoreactivity of proteins in the 50-65 kDa range of molecular weight is mainly observed in the transitional and pre-corneous layers of crocodilians, chelonian, and in the forming beta-layer of lepidosaurians. The ultrastructural localization of the enzyme by immunogold in lizard epidermis during renewal and resting stages shows that the labeling is mainly distributed in the cytoplasm and along the accumulating beta-packets of differentiating beta-cells while it appears very low to undetectable in differentiating alpha-cells of the lacunar, clear, mesos, and alpha-layers. The labeling however becomes absent or undetectable also in the fully mature beta-layer. The study shows that an oxidative enzyme is likely responsible of the cross-linking of the numerous cysteines present in the main proteins accumulated in corneocytes of reptilian epidermis, known as corneous beta-proteins (beta-keratins). This process of disulphide bond formation is probably largely responsible for the formation of hard beta-corneous layers in reptilian scales, a difference with alpha-corneous layers where substrate proteins of transglutaminase appear predominant.
Among vertebrate gastrointestinal microbiome studies, complete representation of taxa is limited, particularly among reptiles. Here, we provide evidence for previously unrecognized host-microbiome associations along the gastrointestinal tract from the American alligator, a crown archosaur with shared ancestry to extinct taxa, including dinosaurs. Microbiome compositional variations reveal that the digestive system consists of multiple, longitudinally heterogeneous microbiomes that strongly correlate to specific gastrointestinal tract organs, regardless of rearing histories or feeding status. A core alligator gut microbiome comprised of Fusobacteria, but depleted in Bacteroidetes and Proteobacteria common to mammalians, is compositionally unique from other vertebrate gut microbiomes, including other reptiles, fish, and herbivorous and carnivorous mammals. As such, modern alligator gut microbiomes advance our understanding of archosaur gut microbiome evolution, particularly if conserved host ecology has retained archosaur-specific symbioses over geologic time.
Minimising stress in farmed crocodiles is not only important for improving animal welfare, but may also improve skin blemish healing and infection resistance, which influence the quality of the final skin product. Forty near-harvest size saltwater crocodiles (1.6-1.8 m TL) from two Australian farms were sampled to evaluate the effect of different pen types (communal pens n=20; individual pens n=20) on stress as indicated by plasma corticosterone. Blood samples were taken within three minutes of immobilisation and analysed using a commercial enzyme immunoassay kit. There was no relationship with animal size (P=0.16), between farms (P=0.86), pen types (P=0.69), communal pens between farms (P=0.28) or individual pens between farms (P=0.24). Based on corticosterone levels, it appears that individual pens do not cause significantly more stress on harvest-size animals than communal pens. Individual pens meet their design specifications by achieving comparable healing rates of belly skin blemishes as communal pens without compromising animal welfare and minimising the possibility of new blemishes.
As part of a larger investigation into three emerging disease syndromes highlighted by conjunctivitis and pharyngitis, systemic lymphoid proliferation and encephalitis, and lymphonodular skin infiltrates in farmed saltwater crocodiles (Crocodylus porosus) and one emerging syndrome of systemic lymphoid proliferation in captive freshwater crocodiles (Crocodylus johnstoni), cytopathic effects (CPE), including syncytial cell formation, were observed in primary crocodile cell lines exposed to clarified tissue homogenates from affected crocodiles. Ten cell cultures with CPE were then screened for herpesviruses using two broadly-reactive herpesvirus PCRs. Amplicons were obtained from 9 of 10 cell cultures and were sequenced. Three novel herpesviruses were discovered and the phylogenetic analysis of these viruses showed there was a 63% Bayesian posterior probability value supporting these viruses clustering with the subfamily Alphaherpesvirinae, and 100% posterior probability of clustering with a clade containing the Alphaherpesvirinae and other unassigned reptile herpesviruses. It is proposed that they are named Crocodyline herpesvirus (CrHV) 1, 2 and 3. CrHV1 and 2 were only isolated from saltwater crocodiles and CrHV3 was only isolated from freshwater crocodiles. A duplex PCR was designed that was able to detect these herpesviruses in formalin-fixed paraffin-embedded tissues, a sample type that neither of the broadly-reactive PCRs was able to detect these herpesviruses in. This work describes the isolation, molecular detection and phylogeny of these novel herpesviruses but the association that they have with the emerging disease syndromes requires further investigation.
Vertebrates coexist with microorganisms in diverse symbiotic associations that range from beneficial to detrimental to the host. Most research has aimed at deciphering the nature of the composite microbial assemblage's genome, or microbiome, from the gastrointestinal (GI) tract and skin of mammals (i.e., humans). In mammals, the GI tract's microbiome aids digestion, enhances uptake of nutrients, and prevents the establishment of pathogenic microorganisms. However, because the GI tract microbiome of the American alligator (Alligator mississippiensis) is distinct from that of all other vertebrates studied to date, being comprised of Fusobacteria in the lower GI tract with lesser abundances of Firmicutes, Proteobacteria, and Bacteroidetes, the function of these assemblages is largely unknown. This review provides a synthesis of our current understanding of the composition of alligators' microbiomes, highlights the potential role of microbiome members in alligators' health (the good), and presents a brief summary of microorganisms detrimental to alligators' health (the bad) including Salmonella spp. and others. Microbial assemblages of the GI tract have co-evolved with their vertebrate host over geologic time, which means that evolutionary hypotheses can be tested using information about the microbiome. For reptiles and amphibians, the number of taxa studied at present is limited, thereby restricting evolutionary insights. Nevertheless, we present a compilation of our current understanding of reptiles' and amphibians' microbiomes, and highlight future avenues of research (the unknown). As in humans, composition of microbiome assemblages provides a promising tool for assessing hosts' health or disease. By further exploring present-day associations between symbiotic microorganisms in the microbiomes of reptiles and amphibians, we can better identify good (beneficial) and bad (detrimental) microorganisms, and unravel the evolutionary history of the acquisition of microbiomes by these poorly-studied vertebrates.
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