Nucleic Acid Preservation Card Surveillance Is Effective for Monitoring Arbovirus Transmission on Crocodile Farms and Provides a One Health Benefit to Northern Australia

The Kunjin strain of West Nile virus (WNV_KUN) is a mosquito-transmitted flavivirus that can infect farmed saltwater crocodiles in Australia and cause skin lesions that devalue the hides of harvested animals. We implemented a surveillance system using honey-baited nucleic acid preservation cards to monitor WNV_KUN and another endemic flavivirus pathogen, Murray Valley encephalitis virus (MVEV), on crocodile farms in northern Australia. The traps were set between February 2018 and July 2020 on three crocodile farms in Darwin (Northern Territory) and one in Cairns (North Queensland) at fortnightly intervals with reduced trapping during the winter months. WNV_KUN RNA was detected on all three crocodile farms near Darwin, predominantly between March and May of each year. Two of the NT crocodile farms also yielded the detection of MVE viral RNA sporadically spread between April and November in 2018 and 2020. In contrast, no viral RNA was detected on crocodile farms in Cairns during the entire trapping period. The detection of WNV_KUN and MVEV transmission by FTA^TM cards on farms in the Northern Territory generally correlated with the detection of their transmission to sentinel chicken flocks in nearby localities around Darwin as part of a separate public health surveillance program. While no isolates of WNV_KUN or MVEV were obtained from mosquitoes collected on Darwin crocodile farms immediately following the FTA^TM card detections, we did isolate another flavivirus, Kokobera virus (KOKV), from Culex annulirostris mosquitoes. Our studies support the use of the FTA^TM card system as a sensitive and accurate method to monitor the transmission of WNV_KUN and other arboviruses on crocodile farms to enable the timely implementation of mosquito control measures. Our detection of MVEV transmission and isolation of KOKV from mosquitoes also warrants further investigation of their potential role in causing diseases in crocodiles and highlights a “One Health” issue concerning arbovirus transmission to crocodile farm workers. In this context, the introduction of FTA^TM cards onto crocodile farms appears to provide an additional surveillance tool to detect arbovirus transmission in the Darwin region, allowing for a more timely intervention of vector control by relevant authorities.     

Characterization of outer ring iodothyronine deiodinases in tissues of the saltwater crocodile (Crocodylus porosus)

The distribution and characterization of outer ring deiodination (ORD) using reverse triiodothyronine (rT3) and thyroxine (T4) as substrates is reported in microsomes of liver, kidney, lung, heart, gut, and brain tissues from juvenile saltwater crocodiles (Crocodylus porosus). In lung and heart only small amounts of rT3 ORD and T4 ORD were detected, while in brain only a small amount of T4 ORD was detected. More detailed characterization studies could be performed on liver, kidney, and gut microsomes. Reverse T3 outer ring deiodination (rT3 ORD) was the predominant activity in liver and kidney microsomes. The properties of crocodile liver and kidney rT3 ORD, such as preference for rT3 as substrate, a dithiothreitol (DTT) requirement of 10 mM, inhibition by propylthiouracil (PTU), and Michaelis-Menten (Km) constant in the micromolar range, correspond to the properties previously reported for a type I deiodinase. The temperature optimum for rT3 ORD was between 30 and 35 degrees. There was also rT3 ORD activity in gut microsomes, along with what appeared to be a type II-like, low-Km deiodinase with a substrate preference for T4. There was also a small amount of T4 ORD activity in liver and kidney microsomes. Liver T4 ORD, like a type II deiodinase, had a preference for T4 as substrate at low substrate concentrations and a DTT requirement of 15 mM and was insensitive to PTU. However, at high substrate concentrations the predominant activity was of the type I deiodinase nature. T4 ORD in liver had an optimal incubation temperature of 30 to 35 degrees. Gut microsomal T4 ORD was also type II-like at low substrate concentrations and type I-like at high substrate concentrations. Gut T4 ORD had an optimal incubation temperature of 25 to 30 degrees and a DTT requirement of 20 mM DTT. Kidney microsomal T4 ORD had the same optimal temperature and DTT requirement as that in gut microsomes; however, there was no competition by low substrate concentrations. These results suggest that ORD in juvenile saltwater crocodile kidney is most likely exclusively catalyzed by a type I-like deiodinase. Liver and gut ORD, in contrast, is catalyzed by two enzymes, with a predominance of a type I-like deiodinase in liver and a type II-like deiodinase in gut. Low-Km T3 IRD activity could not be detected in any tissues of the juvenile saltwater crocodile.     

An investigation of the antimicrobial and anti-inflammatory activities of crocodile oil

Ethnopharmacological relevance: Crocodile oil has been used by traditional practitioners world-wide to treat microbial infections and inflammatory conditions. However, the scientific rationale behind its use is not completely understood. This study provides an updated fatty acid profile and novel scientific evidence of the antimicrobial and anti-inflammatory properties of crocodile oil, obtained from the Nile crocodile (Crocodylus niloticus), justifying its use by traditional healers. Materials and methods: The fatty acid content of the oil was determined by gas chromatography and the major fatty acids were identified. A microplate method was used to assess activity of the oil against Staphylococcus aureus, Klebsiella pneumoniae and Candida albicans. The anti-inflammatory activity of the oil was assessed by oral administration and topical application, utilising a mouse model of acute croton oil-induced contact dermatitis. Results: Sixteen fatty acids were identified with oleic, palmitic and linoleic acid being the major components of the oil. The optimal activity of the oil against the bacteria and fungus was obtained with 15% and 6% (w/v) oil respectively. No significant selectivity was observed against the bacterial species, but Candida albicans was more susceptible. The anti-inflammatory assays showed optimal activity at 3h after the oral administration of oil (60.8±5.5%) and at 12h after topical application (57.5±5.9%). This suggested a short duration of action when the oil was orally administered, and a longer duration of action when it was topically applied. Conclusions: Subsequent studies may be directed towards the investigation of the mechanisms of action of the antimicrobial and anti-inflammatory activities of crocodile oil and its fatty acids.     

Muscle forces acting on the greater trochanter lead to a dorsal warping of the apophyseal growth plate

The apophyseal growth plate of the greater trochanter, unlike most other growth plates of the human body, exhibits a curved morphology that results in a divergent pattern resembling an open crocodile mouth on plain antero-posterior radiographs. To quantify the angular alignment of the growth plate and to draw conclusions about the function of the muscles surrounding it, we analyzed 57 MRI images of 51 children and adolescents aged 3–17 years and of six adults aged 18–52 years. We measured the angulation of the plate relative to the horizontal plane (AY angle) and the trajectories of the muscles attaching to the greater trochanter of the proximal femur. From anterior to posterior, the AY angle shows a decrease of 33.44°. In the anterior third, the cartilage is angled at a mean of 51.64°, and in the posterior third, the mean angulation is 18.6°. This indicates that the cartilage in the anterior region of the greater trochanteric apophysis is subject to more vertically oriented force vectors compared to the posterior region, as the growth plates align perpendicular to the force vectors acting on them. Combining the measured muscle trajectories with the physiological cross-sectional areas (PCSA) available from the literature revealed that, in addition to the known internal and external lateral traction ligament systems, a third, dorsally located traction ligament system exists that may be responsible for the dorsal deformation of the AY angle.     

In vivo confocal microscopy of megalocornea with central mosaic dystrophy

In vivo confocal microscopy was performed on the central cornea of both eyes of a patient with megalocornea and central mosaic dystrophy. In the stroma, starting just below Bowman's membrane, polygonal, moderately reflective areas of opacification separated by diagonal hyporeflective striations were observed. The opaque areas appeared smaller in the anterior stromal layers and seemed to get larger towards the posterior stroma. The epithelium, Bowman's membrane and endothelium appeared normal. In vivo confocal microscopy is helpful in evaluating the morphological characteristics of corneal dystrophies, degenerations or developmental abnormalities of the cornea, and may prove to be helpful in understanding their pathophysiology.     

Thyroid hormone deiodinases during embryonic development of the saltwater crocodile (Crocodylus porosus)

All tissues of the embryonic saltwater crocodile (Crocodylus porosus) gradually increased in weight during development except for lung tissue, which had a peak weight of 1.09 g at day 67, thereafter decreasing in weight. The brain was a relatively large organ. Deiodinase activities in liver, kidney, lung, heart, gut, and brain from day 29 to day 77 of development of the saltwater crocodile were investigated. High-K(m) reverse triiodothyronine (rT(3)) outer ring deiodination (ORD) activity was present in all tissues except the brain. Activity ranged from 559 +/- 51.3 pmol rT(3) deiodinated/mg protein/min in the liver at day 77 to below 10 pmol rT(3) deiodinated/mg protein/min in gut, lung, and heart tissue. rT(3) ORD increased during development in the liver and kidney but decreased in the gut and lung. Activity in the heart was very low (less than 2 pmol rT(3) deiodinated/mg protein/min) and did not change during development. Low-K(m) thyroxine (T(4)) ORD in liver and kidney tissue had peaks of activity around day 49 of incubation (0.52 and 0.09 fmol T(4) deiodinated/mg protein/min, respectively). After day 49, T(4) ORD activity in these tissues decreased. T(4) ORD activity in gut, lung, and heart was very low (less than 0.04 fmol T(4) deiodinated/mg protein/min), with activity in lung increasing slightly during the rest of development. T(4) ORD activity in the brain increased toward day 77 (0.14 +/- 0.03 fmol T(4) deiodinated/mg protein/min), illustrating its importance in local triiodothyronine (T(3)) production during brain development. T(3) inner ring deiodination activity was present only in the embryonic liver and peaked at day 49 (10.1 fmol T(3) deiodinated/mg protein/min), after which activity decreased.     

Connection of the Posterior Occipital Muscle and Dura Mater of the Siamese Crocodile

The myodural bridge was proposed initially in 1995. The myodural bridge is a connective tissue bridge that connects a pair of deep muscles at the suboccipital region to the dura mater. There have been numerous studies concerning the morphology and function of the myodural bridge. To determine whether a myodural bridge exists in reptiles, six Siamese crocodiles were investigated using gross anatomy dissection and P45 sheet plastination technologies. As a result, we demonstrated that the posterior occipital muscles of the Siamese crocodile are directly or indirectly connected to the proatlas, atlas, and intermembrane between them. Multiple trabeculae existing in the posterior epidural space extended from the ventral surface of the proatlas, atlas, and intermembrane between them to the dorsal surface of the spinal dura mater. This study showed that the posterior occipital muscle in the suboccipital region of the Siamese crocodile is connected to the spinal dura mater through the proatlas, atlas, and the trabeculae. In conclusion, a myodural bridge‐like structure exists in reptiles. This connection may act as a pump to provide cerebrospinal fluid (CSF) circulation at the occipitocervical junction. We hypothesize that a physiologic role of the Siamese crocodile's myodural bridge may be analogous to the human myodural bridge. Anat Rec, 299:1402–1408, 2016. © 2016 Wiley Periodicals, Inc.     

鳄鱼血血红蛋白大小随浓度、温度和pH的变化

目的：应用激光散射技术对鳄鱼血血红蛋白的大小及粒度分布等特性进行系统性测定分析,并研究其在不同温度、浓度及pH值下的变化规律。方法：用激光散射装置测定不同浓度、温度和pH值下的鳄鱼血红蛋白的粒度及粒度分布。结果：①鳄鱼血红蛋白的粒径基本不随其浓度变化,不会发生解聚或聚集的情况。②鳄鱼血红蛋白粒径在10℃～48℃之间基本不变,但在高温时（≥48℃）会发生聚集而使其粒径变大一倍。③鳄鱼血红蛋白耐碱,其粒径从生理pH升到9.21都不会发生变化。但在酸性的情况下会发生聚集粒径变大一倍。结论：①鳄鱼血红蛋白的粒径在不同Hb浓度下基本保持不变,说明其结构比较稳定,在不同浓度下都不会发生聚集或解聚现象。②鳄鱼血红蛋白在10℃～48℃的条件下,其粒径都没有变化,说明了血红蛋白的结构对于温度来说也是相当稳定的。③鳄鱼血红蛋白在正常pH至高碱性环境下,其粒径也基本不会变化。说明了鳄鱼血红蛋白在碱性的环境下其稳定性很好,不会发生聚集或解聚的情况。