Search Results (11)

Saltwater crocodile (SC; Crocodylus porosus) bone, an underutilized by-product, can be converted into high-value bio-calcium (Biocal), serving as a potential source of calcium and minerals. This study aimed to produce SC bone Biocal as functional gel enhancer for fish bologna development and to increase calcium intake. The resulting bone powder was evaluated for physicochemical, microbiological, and molecular properties. Additionally, the textural, physicochemical, structural, and sensorial properties of the formulated fish bologna incorporating Biocal at varying levels (0–10% w/w) were also evaluated. Biocal, obtained as a fine white powder, had a 16.83% yield. Mineral analysis showed 26.25% calcium and 13.72% phosphorus, with no harmful metals or pathogens detected. X-ray diffraction confirmed hydroxyapatite with 69.92% crystallinity, while calcium bioavailability was measured at 22.30%. Amino acid analysis indicated high levels of glycine, proline, and hydroxyproline, essential for collagen support. The findings confirmed that SC bone Biocal is beneficial and safe for food fortification. Incorporating SC Biocal (2–10% w/w) significantly affected the fish bologna characteristics (p < 0.05). As the Biocal level increased, the gel strength, hardness, and shear force also increased. The addition of 6% (w/w) Biocal significantly improved the textural property, without a detrimental effect on the sensory attributes of the bologna gel (p < 0.05). SDS-PAGE analysis showed TGase-enhanced myosin heavy chain (MHC) cross-linking, particularly in combination with Biocal. Moreover, the enriched Biocal–bologna gel exhibited a finer and denser microstructure. Thus, SC Biocal, particularly at 6% (w/w), can serve as a functional gel enhancer in surimi-based products, without compromising organoleptic quality. Full article

Hybrids between the critically endangered Siamese crocodile (Crocodylus siamensis) and least-concern saltwater crocodile (C. porosus) in captive populations represent a serious challenge for conservation and reintroduction programs due to the impact of anthropogenic activities. A previous study used microsatellite and mitochondrial DNA data to establish the criteria for identifying species and their hybrids; however, the results may have been influenced by biased allelic frequencies and genetic drift within the examined population. To overcome these limitations and identify the true signals of selection, alternative DNA markers and a diverse set of populations should be employed. Therefore, this study used DArT sequencing to identify genome-wide single nucleotide polymorphisms (SNPs) in both species and confirm the genetic scenario of the parental species and their hybrids. A population of saltwater crocodiles from Australia was used to compare the distribution of species-diagnostic SNPs. Different analytical approaches were compared to diagnose the level of hybridization when an admixture was present, wherein three individuals had potential backcrossing. Approximately 17.00–26.00% of loci were conserved between the Siamese and saltwater crocodile genomes. Species-diagnostic SNP loci for Siamese and saltwater crocodiles were identified as 8051 loci and 1288 loci, respectively. To validate the species-diagnostic SNP loci, a PCR-based approach was used by selecting 20 SNP loci for PCR primer design, among which 3 loci were successfully able to differentiate the actual species and different hybridization levels. Mitochondrial and nuclear genetic information, including microsatellite genotyping and species-diagnostic DNA markers, were combined as a novel method that can compensate for the limitations of each method. This method enables conservation prioritization before release into the wild, thereby ensuring sustainable genetic integrity for long-term species survival through reintroduction and management programs. Full article

(1) Background: Pancuronium bromide is a neuromuscular blocker used for immobilizing crocodiles that can be reversed with neostigmine. A recommended drug dose has only been established for saltwater crocodiles (Crocodylus porosus), mostly based on trials in juveniles and subadults. After trialing a dose recommendation in a small cohort of nine Nile crocodiles (Crocodylus niloticus), we developed and applied a new dose recommendation for large adult Nile crocodiles. (2) Methods: we trialed and adapted a pancuronium bromide (Pavulon 4 mg/2 mL) dose in Nile crocodiles originally established for saltwater crocodiles and applied the new dose for the immobilization of 32 Nile crocodiles destined for transport. Reversal was achieved with neostigmine (Stigmine 0.5 mg/mL). (3) Results: Nine crocodiles were included in the trial phase; the induction time was highly variable (average: 70 min; range: 20–143 min), and the recovery time was prolonged (average: 22 h; range: 50 min–5 days), especially in large animals after reversal with neostigmine. Based on these results, we established a dose-independent recommendation (3 mg pancuronium bromide and 2.5 mg neostigmine) for animals weighing ≥ 270 kg (TL ≥ ~3.8 m). When applied to 32 adult male crocodiles (BW range: 270–460 kg; TL range: 3.76–4.48 m), the shortest induction time was ~20 min and the longest ~45 min. (4) Conclusions: Pancuronium bromide and its antidote, neostigmine, are effective for the immobilization and reversal of adult male Nile crocodiles (TL ≥ 3.8 m or BW ≥ 270 kg) when given in a weight-independent fashion. Full article

Populations of Siamese crocodiles (Crocodylus siamensis) have severely declined because of hunting and habitat fragmentation, necessitating a reintroduction plan involving commercial captive-bred populations. However, hybridization between Siamese and saltwater crocodiles (C. porosus) has occurred in captivity. Siamese crocodiles commonly have post-occipital scutes (P.O.) with 4–6 scales, but 2–6 P.O. scales were found in captives on Thai farms. Here, the genetic diversity and population structure of Siamese crocodiles with large P.O. variations and saltwater crocodiles were analyzed using mitochondrial DNA D-loop and microsatellite genotyping. Possible crocodile hybrids or phenotypic variations were ascertained by comparison with our previous library from the Siam Crocodile Bioresource Project. Siamese crocodiles with <4 P.O. scales in a row exhibit normal species-level phenotypic variation. This evidence encourages the revised description of Siamese crocodiles. Moreover, the STRUCTURE plot revealed large distinct gene pools, suggesting crocodiles in each farm were derived from distinct lineages. However, combining both genetic approaches provides evidence of introgression for several individual crocodiles, suggesting possible hybridization between Siamese and saltwater crocodiles. We proposed a schematic protocol with patterns observed in phenotypic and molecular data to screen hybrids. Identifying non-hybrid and hybrid individuals is important for long-term in situ/ex situ conservation. Full article

Tandemly arranged and dispersed repetitive DNA sequences are important structural and functional elements that make up a significant portion of vertebrate genomes. Using high throughput, low coverage whole genome sequencing followed by bioinformatics analysis, we have identified seven major tandem repetitive DNAs and two fragments of LTR retrotransposons in the genome of the Nile crocodile (Crocodylus niloticus, 2n = 32). The repeats showed great variability in structure, genomic organization, and chromosomal distribution as revealed by fluorescence in situ hybridization (FISH). We found that centromeric and pericentromeric heterochromatin of C. niloticus is composed of previously described in Crocodylus siamensis CSI-HindIII and CSI-DraI repetitive sequence families, a satellite revealed in Crocodylus porosus, and additionally contains at least three previously unannotated tandem repeats. Both LTR sequences identified here belong to the ERV1 family of endogenous retroviruses. Each pericentromeric region was characterized by a diverse set of repeats, with the exception of chromosome pair 4, in which we found only one type of satellite. Only a few repeats showed non-centromeric signals in addition to their centromeric localization. Mapping of 18S–28S ribosomal RNA genes and telomeric sequences (TTAGGG)_n did not demonstrate any co-localization of these sequences with revealed centromeric and pericentromeric heterochromatic blocks. Full article

The risk of flavivirus infections among the crocodilian species was not recognised until West Nile virus (WNV) was introduced into the Americas. The first outbreaks caused death and substantial economic losses in the alligator farming industry. Several other WNV disease episodes have been reported in crocodilians in other parts of the world, including Australia and Africa. Considering that WNV shares vectors with other flaviviruses, crocodilians are highly likely to also be exposed to flaviviruses other than WNV. A serological survey for flaviviral infections was conducted on saltwater crocodiles (Crocodylus porosus) at farms in the Northern Territory, Australia. Five hundred serum samples, collected from three crocodile farms, were screened using a pan-flavivirus-specific blocking ELISA. The screening revealed that 26% (n = 130/500) of the animals had antibodies to flaviviruses. Of these, 31.5% had neutralising antibodies to WNV_KUN (Kunjin strain), while 1.5% had neutralising antibodies to another important flavivirus pathogen, Murray Valley encephalitis virus (MVEV). Of the other flaviviruses tested for, Fitzroy River virus (FRV) was the most frequent (58.5%) in which virus neutralising antibodies were detected. Our data indicate that farmed crocodiles in the Northern Territory are exposed to a range of potentially zoonotic flaviviruses, in addition to WNV_KUN. While these flaviviruses do not cause any known diseases in crocodiles, there is a need to investigate whether infected saltwater crocodiles can develop a viremia to sustain the transmission cycle or farmed crocodilians can be used as sentinels to monitor the dynamics of arboviral infections in tropical areas. Full article

We have previously found that sera from Crocodylus porosus contain anticancer agents and the treatment of MCF7 cells with this serum resulted in the differential expression of 51 genes. The purpose of this study was to use in silico analysis to identify genes that might be epigenetically modulated in cells treated with crocodile serum and to understand the role of potential genes as novel candidates with epigenetic therapeutic potential. The findings report five proto-oncogenes (TUBA1B, SLC2A1, PGK1, CCND1, and NCAPD2) and two tumor suppressor genes (RPLP2, RPL37) as novel therapeutic targets. Furthermore, we present a comprehensive overview of relevant studies on epigenetic regulation of these genes along with an insight into their clinical implications. Therefore, elucidating the molecules present in the serum and gut bacteria of reptiles such as crocodiles may offer insights into the role of these genes on longevity, health, disease, and life expectancy. Full article

Crocodiles are remarkable animals that have the ability to endure extremely harsh conditions and can survive up to a 100 years while being exposed to noxious agents that are detrimental to Homo sapiens. Besides their immunity, we postulate that the microbial gut flora of crocodiles may produce substances with protective effects. In this study, we isolated and characterized selected bacteria colonizing the gastrointestinal tract of Crocodylusporosus and demonstrated their inhibitory effects against three different cancerous cell lineages. Using liquid chromatography-mass spectrometry, several molecules were identified. For the first time, we report partial analyses of crocodile’s gut bacterial molecules. Full article

West Nile virus, Kunjin strain (WNV_KUN) is endemic in Northern Australia, but rarely causes clinical disease in humans and horses. Recently, WNV_KUN genomic material was detected in cutaneous lesions of farmed saltwater crocodiles (Crocodylus porosus), but live virus could not be isolated, begging the question of the pathogenesis of these lesions. Crocodile hatchlings were experimentally infected with either 10⁵ (n = 10) or 10⁴ (n = 11) TCID₅₀-doses of WNV_KUN and each group co-housed with six uninfected hatchlings in a mosquito-free facility. Seven hatchlings were mock-infected and housed separately. Each crocodile was rotationally examined and blood-sampled every third day over a 3-week period. Eleven animals, including three crocodiles developing typical skin lesions, were culled and sampled 21 days post-infection (dpi). The remaining hatchlings were blood-sampled fortnightly until experimental endpoint 87 dpi. All hatchlings remained free of overt clinical disease, apart from skin lesions, throughout the experiment. Viremia was detected by qRT-PCR in infected animals during 2–17 dpi and in-contact animals 11–21 dpi, indicating horizontal mosquito-independent transmission. Detection of viral genome in tank-water as well as oral and cloacal swabs, collected on multiple days, suggests that shedding into pen-water and subsequent mucosal infection is the most likely route. All inoculated animals and some in-contact animals developed virus-neutralizing antibodies detectable from 17 dpi. Virus-neutralizing antibody titers continued to increase in exposed animals until the experimental endpoint, suggestive of persisting viral antigen. However, no viral antigen was detected by immunohistochemistry in any tissue sample, including from skin and intestine. While this study confirmed that infection of saltwater crocodiles with WNV_KUN was associated with the formation of skin lesions, we were unable to elucidate the pathogenesis of these lesions or the nidus of viral persistence. Our results nevertheless suggest that prevention of WNV_KUN infection and induction of skin lesions in farmed crocodiles may require management of both mosquito-borne and water-borne viral transmission in addition to vaccination strategies. Full article

Saltwater crocodilepox virus (SwCRV), belonging to the genus Crocodylidpoxvirus, are large DNA viruses posing an economic risk to Australian saltwater crocodile (Crocodylus porosus) farms by extending production times. Although poxvirus-like particles and sequences have been confirmed, their infection dynamics, inter-farm genetic variability and evolutionary relationships remain largely unknown. In this study, a poxvirus infection dynamics study was conducted on two C. porosus farms. One farm (Farm 2) showed twice the infection rate, and more concerningly, an increase in the number of early- to late-stage poxvirus lesions as crocodiles approached harvest size, reflecting the extended production periods observed on this farm. To determine if there was a genetic basis for this difference, 14 complete SwCRV genomes were isolated from lesions sourced from five Australian farms. They encompassed all the conserved genes when compared to the two previously reported SwCRV genomes and fell within three major clades. Farm 2′s SwCRV sequences were distributed across all three clades, highlighting the likely mode of inter-farm transmission. Twenty-four recombination events were detected, with one recombination event resulting in consistent fragmentation of the P4c gene in the majority of the Farm 2 SwCRV isolates. Further investigation into the evolution of poxvirus infection in farmed crocodiles may offer valuable insights in evolution of this viral family and afford the opportunity to obtain crucial information into natural viral selection processes in an in vivo setting. Full article

The role that oil palm plays in the Lower Kinabatangan region of Eastern Sabah is of considerable scientific and conservation interest, providing a model habitat for many tropical regions as they become increasingly fragmented. Crocodilians, as apex predators, widely distributed throughout the tropics, are ideal indicator species for ecosystem health. Drones (or unmanned aerial vehicles (UAVs)) were used to identify crocodile nests in a fragmented landscape. Flights were targeted through the use of fuzzy overlay models and nests located primarily in areas indicated as suitable habitat. Nests displayed a number of similarities in terms of habitat characteristics allowing for refined modelling of survey locations. As well as being more cost-effective compared to traditional methods of nesting survey, the use of drones also enabled a larger survey area to be completed albeit with a limited number of flights. The study provides a methodology for targeted nest surveying, as well as a low-cost repeatable flight methodology. This approach has potential for widespread applicability across a range of species and for a variety of study designs. Full article