An Immunohistochemical Approach to Identify the Sex of Young Marine Turtles

Marine turtles exhibit temperature‐dependent sex determination (TSD). During critical periods of embryonic development, the nest's thermal environment directs whether an embryo will develop as a male or female. At warmer sand temperatures, nests tend to produce female‐biased sex ratios. The rapid increase of global temperature highlights the need for a clear assessment of its effects on sea turtle sex ratios. However, estimating hatchling sex ratios at rookeries remains imprecise due to the lack of sexual dimorphism in young marine turtles. We rely mainly upon laparoscopic procedures to verify hatchling sex; however, in some species, morphological sex can be ambiguous even at the histological level. Recent studies using immunohistochemical (IHC) techniques identified that embryonic snapping turtle (Chelydra serpentina) ovaries overexpressed a particular cold‐induced RNA‐binding protein in comparison to testes. This feature allows the identification of females vs. males. We modified this technique to successfully identify the sexes of loggerhead sea turtle (Caretta caretta) hatchlings, and independently confirmed the results by standard histological and laparoscopic methods that reliably identify sex in this species. We next tested the CIRBP IHC method on gonad samples from leatherback turtles (Dermochelys coriacea). Leatherbacks display delayed gonad differentiation, when compared to other sea turtles, making hatchling gonads difficult to sex using standard H&E stain histology. The IHC approach was successful in both C. caretta and D. coriacea samples, offering a much‐needed tool to establish baseline hatchling sex ratios, particularly for assessing impacts of climate change effects on leatherback turtle hatchlings and sea turtle demographics. Anat Rec, 300:1512–1518, 2017. © 2017 Wiley Periodicals, Inc.     

Osteological Histology of the Pan‐Alcidae (Aves,Charadriiformes): Correlates of Wing‐Propelled Diving and Flightlessness

Although studies of osteological morphology, gross myology, myological histology, neuroanatomy, and wing‐scaling have all documented anatomical modifications associated with wing‐propelled diving, the osteohistological study of this highly derived method of locomotion has been limited to penguins. Herein we present the first osteohistological study of the derived forelimbs and hind limbs of wing‐propelled diving Pan‐Alcidae (Aves, Charadriiformes). In addition to detailing differences between wing‐propelled diving charadriiforms and nondiving charadriiforms, microstructural modifications to the humeri, ulnae and femora of extinct flightless pan‐alcids are contrasted with those of volant alcids. Histological thin‐sections of four species of pan‐alcids (Alca torda, ?Alca grandis, ?Pinguinus impennis, ?Mancalla cedrosensis) and one outgroup charadriiform (Stercorarius longicaudus) were compared. The forelimb bones of wing‐propelled diving charadriiforms were found to have significantly thicker (～22%) cortical bone walls. Additionally, as in penguins, the forelimbs of flightless pan‐alcids are found to be osteosclerotic. However, unlike the pattern documented in penguins that display thickened cortices in both forelimbs and hind limbs, the forelimb and hind limb elements of pan‐alcids display contrasting microstructural morphologies with thickened forelimb cortices and relatively thinner femoral cortices. Additionally, the identification of medullary bone in the sampled ?Pinguinus impennis specimen suggests that further osteohistological investigation could provide an answer to longstanding questions regarding sexual dimorphism of Great Auks. Finally, these results suggest that it is possible to discern volant from flightless wing‐propelled divers from fragmentary fossil remains. Anat Rec, 297:188–199, 2014. © 2013 Wiley Periodicals, Inc.     

<Emphasis Type="Italic">Camallanus</Emphasis> Railliet et Henry, 1915 (Nematoda,Camallanidae) from Australian freshwater turtles with descriptions of two new species and molecular differentiation of known taxa

Two new species of Camallanus are described from Australian freshwater turtles. Camallanus beveridgei sp. nov. is reported from Elseya dentata in Northern Territory. It differs from other species of the genus parasitic in turtles by several characters including the shape of the median ridge in the buccal capsule and the position of the anterior pair of caudal papillae in males. Camallanus sprenti sp. nov. is reported from Elseya latisternum (type host) and Emydura krefftii in northern Queensland. It is closely related to Camallanus tuckeri, and differs from the latter species in possessing a shorter oesophagus. We summarize data on morphology, distribution and specificity of 5 known Camallanus spp. from Australian turtles and provide a key for their identification. Sequence comparison of more than 500 base pairs at the 5′ end of the nuclear 28S rDNA gene confirms the status of C. sprenti and C. beveridgei as new species. Camallanus sprenti differs from the other 4 species of Camallanus from Australian turtles by 16–59 bases (3.1–11.5%) while C. beveridgei differed from the other 4 species by 23–60 bases (4.5–11.6%). Phylogenetic analysis demonstrates close interrelationships among C. tuckeri, C. sprenti and C. beveridgei, the three species with most similar buccal capsules.     

Osteohistological correlates of muscular attachment in terrestrial and freshwater Testudines

Sharpey's fibers are considered the anatomical structures integrated to the muscles. Since these fibers leave marks at the microscopic level, their presence and distribution are used as evidence of muscle attachment in extinct and extant forms. In recent years, studies have been focusing on muscle–bone and tendon–bone interaction mostly on mammals. The main objective of this work is to contribute to the morphological and histological knowledge of muscle attachment in other amniotes, such as reptiles, and their variation related to different locomotor habits. In this way, a study was performed on terrestrial and aquatic turtles. The musculature related to the movement of the humerus, and pectoral girdle in Chelonoidis chilensis, Phrynops hilarii and Hydromedusa tectifera was analyzed. Dissections were performed mapping the origins and insertions of each muscle and undecalcified thin sections were performed in specific muscular attachment sites. We found some differences which were not previously reported, related to the insertion of the m. pectoralis, the m. coracobrachialis magnus and the origin of the m. tractor radii. The osteohistology revealed the presence of Sharpey's fibers in the cortex of all the bone elements analyzed. Patterns were established in relation to the orientation and density of Sharpey's fibers, which were used for the categorization of each muscle attachment site. The comparative micro‐anatomical study of these areas did not reveal any important differences between terrestrial and freshwater turtles in muscles involved with the rotation, abduction and adduction of the humerus. In this way, the preliminary results suggest an absence of correlation between the distribution and density of Sharpey's fibers between different habitat forms, at least in the bones and species analyzed.     

Development of a polymerase chain reaction assay for specific identification of Clostridium colinum

Clostridium colinum is the causative agent of ulcerative enteritis, a serious disease of the bobwhite quail (Colinus virginianus) and sporadically of young chickens. The aim of the present study was to develop a polymerase chain reaction (PCR) assay specific for C. colinum identification. The 16S rDNA sequence of C. colinum was analysed and two species-specific primers were designed. The specificity of these primers was tested with closely related Clostridium species and the expected amplified product (935 base pairs) was observed only with DNA from samples containing C. colinum. Results from performing PCR assays on faecal samples from quails spiked with different concentrations of C. colinum, showed that the detection limit of the assay was 1.6×10⁴ colony-forming units per gram of faecal material. This PCR assay can be used in diagnostic laboratories to confirm the presence of C. colinum in pure cultures and could be used to screen enriched samples or faecal samples for the presence of this pathogen.     

Sequencing and characterization of the full-length gene encoding the single-stranded DNA binding protein of a novel <Emphasis Type="Italic">Chelonian</Emphasis> herpesvirus

Summary. Through 4 consecutive genomic walks employing a recently modified inverse polymerase chain reaction technique, a 4,054-bp DNA fragment of a newfound green turtle herpesvirus (GTHV) was obtained from tumor tissues of a green turtle with fibropapillomas. This newly identified viral DNA fragment contains two non-overlapping open reading frames (ORF) oriented in the 3 to 5 direction. The first ORF is 59% G+C rich and contains the full-length genomic sequence of the DNA binding protein (DBP) gene (3,585bp) encoding a protein of 1,195 amino acid residues in length. The second ORF encodes a partial peptide of the UL28 gene. Phylogenetic analysis of the GTHV DBP gene confirmed and substantiated that this novel Chelonian herpesvirus is closely related to the subfamily Alphaherpesvirinae. Examination of the translated amino acid sequence further supports this categorization since GTHV DBP comprises a highly conserved zinc finger motif (CXLCX₄RX₂C) and a putative DNA binding domain, and exhibits high sequence homology to other alphaherpesviruses. Cloning and sequencing the genome of this putative herpesvirus will facilitate current understanding of its role in causing GTFP and the development of molecular- and immuno-based methods for the diagnosis and prevention of this devastating disease of green turtles.     

Reassessment of histopathology and dermoscopy findings in 145 Japanese cases of melanocytic nevus of the sole: Toward a pathological diagnosis of early‐stage malignant melanoma in situ

Recently, dermoscopic visualization has been improved, allowing for the identification of malignant melanoma (MM) of the sole in situ. When the parallel ridge pattern is evident on dermoscopy, the proliferation of solitarily arranged melanocytes in the crista profunda intermedia should be examined histologically, since this may be a clue to the early diagnosis of MM in situ. We reviewed 145 Japanese cases of melanocytic nevus on the sole, and investigated several useful histological features for the diagnosis of MM in situ using a recent proposal as well as several standard histological criteria of MM in situ. Five cases were considered to be an early‐stage MM in situ out of 145 cases previously diagnosed as melanocytic nevi of the sole. These cases showed several specific features, including solitarily arranged melanocytes or melanocyte nests comprising fewer than four cells. Our findings indicate that early‐stage MM of the sole in situ can be diagnosed by using new dermoscopy‐related histological findings. They are (i) irregular distribution of solitary melanocytes at the crista profunda intermedia with or without small nests (up to three melanocytes) on the slope of rete ridges; and (ii) larger melanocytes with a halo around the nucleus.     

Recessive mutations in RYR1 are a common cause of congenital fiber type disproportion

The main histological abnormality in congenital fiber type disproportion (CFTD) is hypotrophy of type 1 (slow twitch) fibers compared to type 2 (fast twitch) fibers. To investigate whether mutations in RYR1 are a cause of CFTD we sequenced RYR1 in seven CFTD families in whom the other known causes of CFTD had been excluded. We identified compound heterozygous changes in the RYR1 gene in four families (five patients), consistent with autosomal recessive inheritance. Three out of five patients had ophthalmoplegia, which may be the most specific clinical indication of mutations in RYR1. Type 1 fibers were at least 50% smaller, on average, than type 2 fibers in all biopsies. Recessive mutations in RYR1are a relatively common causeof CFTD and can be associated with extreme fiber size disproportion. © 2010 Wiley‐Liss, Inc.     

Computed tomographic analysis of internal structures within the nasal cavities of green,loggerhead and leatherback sea turtles

The morphology of the tetrapod nasal cavity has adapted to the environment in terms of olfaction and respiration. Reports indicate that the internal structure of the nasal cavity of green sea turtles is more complex than that of turtles in general, but whether or not it is similar among sea turtle species remains unknown. The present study aimed to define the internal structures of the nasal cavity of green (Chelonian mydas), loggerhead (Caretta caretta) and leatherback (Dermochelys coriacea) sea turtles using computed tomography. The nasal cavity of green and loggerhead sea turtles contained anterodorsal, anteroventral, posterodorsal diverticula and a posteroventral excavation in the middle. In contrast, the nasal cavity of leatherback sea turtles had more complicated dorsal region comprising anterodorsal and posterodorsal diverticula, and two excavations between the nostril and anterodorsal diverticulum, but no distinct structures at the ventral region. The airway in the nasal cavity was shorter and thicker in the leatherback, than in the green and loggerhead turtles. These species differences might reflect ecological variety and different evolutionary strategies.     

Nematode parasites of Chelidae (Testudines) from Southern Brazil

The presence of helminths associated with freshwater turtles is rarely reported. There are no records of nematodes parasitizing Acanthochelys spixii, and for Hydromedusa tectifera, there is only the report of unidentified nematodes found in this species in Argentina. This is the first report of nematodes (Spiroxys contortus and Camallanus sp.) in A. spixii and the first record of Spiroxys contortus and Camallanus sp. in H. tectifera. This is the southernmost record of S . contortus because this nematode was previously recorded only in Mexico.     

A chicken intestinal ligated loop model to study the virulence of Clostridium perfringens isolates recovered from antibiotic-free chicken flocks

Necrotic enteritis (NE) is a major problem in antibiotic-free (ABF) chicken flocks and specific strains of Clostridium perfringens are known to induce NE. The objective of this study was to develop a chicken intestinal ligated loop model in order to compare the virulence of various C. perfringens strains recovered from consecutive ABF flocks with and without NE. Intestinal loops were surgically prepared in 10 anaesthetized specific-pathogen-free chickens and alternately inoculated with C. perfringens isolates or brain heart infusion (BHI) media. Histological lesion scoring was performed for each loop. All strains from NE-affected flocks induced histological lesions compatible with NE whereas inoculation of loops with a commensal C. perfringens strain or BHI did not. Among inoculated strains, CP0994 (netB-positive and cpb2-positive) and CP-2003-1256 (netB-positive) demonstrated mean histological lesion scores significantly higher (P?netB-negative and cpb2-positive) induced intestinal lesions without significantly higher scores. In loops where villi were colonized by Gram-positive rods, significantly higher (P?C. perfringens is a critical step in the pathogenesis of NE. Finally, we demonstrated the importance of controlling virulent C. perfringens strains in ABF chicken flocks as a highly virulent strain can be present in consecutive flocks with NE and possibly affect multiple flocks.     

Distribution of MED12 mutations in fibroadenomas and phyllodes tumors of the breast—implications for tumor biology and pathological diagnosis

Somatic mutations in exon 2 of MED12 have been described in benign and malignant smooth muscle cell tumors suggesting a functional role in these neoplasms. Recently fibroadenomas of the breast were also reported to harbor MED12 mutations. Hence, we explored MED12 mutations in fibroepithelial tumors of the breast, histological subtypes of fibroadenomas and phyllodes tumors, to validate and extend previous efforts. Using conventional Sanger sequencing, we profiled 39 cases of fibroepithelial breast tumors comprising classic histological subtypes of fibroadenomas as well as benign and malignant phyllodes tumors for mutations in exon 2 of MED12. MED12 mutations were detected in 60% of all tumor samples with the majority being missense mutations affecting codon 44. Additionally, we report novel in‐frame deletions that have not been described previously. Sixty‐two percent of the fibroadenomas harbored mutated MED12 with intracanalicular fibroadenomas being the most frequently mutated histological subtype (82%). Of note, 8/11 of benign phyllodes tumors had MED12 mutations while only 1/5 of malignant phyllodes tumors showed mutations in exon 2 of MED12. In conclusion, we confirm the frequent occurrence of MED12 mutations in fibroadenomas, provide evidence that most intracanalicular fibroadenomas closely resembling benign phyllodes as well as benign phyllodes tumors harbor MED12 mutations, and conclude that MED12 mutations in malignant phyllodes tumors appear to be relatively rare. © 2015 Wiley Periodicals, Inc.     

Scanning electron microscope studies of the papilla basilaris of some turtles and snakes

The papillae basilares of three species of turtles and four species of snakes were studied by SEM. The papillae of turtles are relatively large among reptiles and are characterized by a long, horizontal middle section resting on a wide basilar membrane. Both terminal ends of the papilla extend onto the surrounding limbus in the form of a forked or “T”-shaped end or as a curved, “hook”-like process. Details vary with the species. In the three species of turtles studied, there were between 1,100 and 1,400 hair cells on a papilla. The tectorial membrane covering the horizontal portion of the papilla is heavy in appearance and tightly attached to the kinocilial bulbs. The terminal ends of the papilla are covered by a thin gelatinous material. In addition, a mat-like tectorial network covers the supporting cells and extends from the microvilli of the supporting cells to the overlying tectorial membrane. All hair cells are unidirectionally and abneurally oriented. The supporting cell surfaces form a large part of the papilla and, thus, hair cell density is low. The papillae of the two boid snake species studied are moderately long among snakes and contain a moderate number of hair cells (574 in Epicrates and 710–780 in Constrictor). Papillar form is elongate, ovoid, or canoe-shaped. The tectorial membrane may be either highly fenestrated or moderately dense and covers all but a few of the terminal hair cells. A tectorial-like mat covers all but a few of the terminal hair cells. Most hair cells are unidirectionally and abneurally oriented. A few terminal cells in boids may show reverse orientation. Hair cell density is similar to that of turtles.     

NetB-producing and beta2-producing Clostridium perfringens associated with subclinical necrotic enteritis in laying hens in the Netherlands

Since 2006 increasing numbers of laying hen flocks with decreased production have been reported in the Netherlands. At necropsy, birds from affected flocks showed multifocal areas of necrosis in the duodenum. Histologically the duodenum had moderate to marked villus atrophy and fusion with crypt hyperplasia and a mixed inflammatory infiltrate within the lamina propria underlying focal areas of degenerative epithelium. Multifocally, free within the intestinal lumen and associated with epithelial necrosis, were marked numbers of large rod-shaped bacteria. Anaerobic culturing and subsequent toxin typing revealed, in 19 out of 73 affected birds, the presence of Clostridium perfringens strains, either type A or type C harbouring the atypical allele of cpb2 and netB. Eighteen out of these 19 birds carried C. perfringens strains capable of producing beta2 toxin in vitro and all of these birds harboured C. perfringens strains capable of producing NetB toxin in vitro. In contrast, specific pathogen free (SPF) birds lacked gross or histological lesions in their duodenum, and C. perfringens type C was isolated from four out of 15 SPF birds tested. One of these isolates harboured the consensus three allele of cpb2 that produced beta2 toxin in vitro. None of the C. perfringens isolates originating from SPF birds harboured netB. These findings might indicate that the NetB toxin produced by C. perfringens is associated with subclinical necrotic enteritis in layers, whereas the involvement of beta2 toxin in subclinical necrotic enteritis, if any, might be variant dependent.     

Effects of different health status on blood haematochemical values of loggerhead sea turtles (Caretta caretta)

In reptiles, several studies show the correlation between good or poor health and changes of physiologic parameters, but the comparison among data, methods and biochemical analysis in sea turtles are difficult. This study was carried out to investigate the biochemical patterns of total protein, albumin, creatinine, urea, alanine aminotransferase and aspartate aminotransferase (AST) in 100 loggerhead sea turtles (52 in good health, 16 with illness and 32 with serious illness). Blood samples were collected by venipuncture from the dorsal cervical sinus, the jugular vein or the ventral caudal sinus between March and October 2007 to 2009. Compared with subjects in good health, total protein and albumin concentrations were lower in subjects suffering from illness (p < 0.001) and in those with serious illness (p < 0.01). Compared with subjects with good health, creatinine concentrations (p < 0.001) and AST activity (p < 0.001) were lower in turtles with serious illness (p < 0.001), and urea concentrations were lower in subjects suffering from (p < 0.01). One-way repeated-measures analysis of variance showed significant effects of the health status of sea turtles on albumin (p < 0.05), urea (p < 0.001) and AST (p < 0.001) fluctuations. Data obtained showed a significant correlation between health status and haematochemical changes in sea turtles.     

Transfer of rye chromosome segments to wheat by a gametocidal system

A gametocidal chromosome derived from Aegilops triuncialis (3C) induces chromosome mutations in gametes lacking the 3C chromosome in common wheat (Triticum aestivum L.). We combined 3C with chromosome 1R of rye (Secale cereale L.) in a common wheat line to know how efficiently 3C induces transfers of small 1R segments to wheat. In the 811 progeny of this wheat line, we found five wheat chromosomes (2A, 2D, 3D, 5D and 7D) carrying segments of the 1R satellite. Wheat plants carrying these translocations were tested for the presence of a storage protein locus Sec-1 and a cluster of resistance genes for wheat rust diseases, Sr31, Lr26 and Yr9. The 2A and 2D translocations had the Sec-1 and three rust resistance loci. The 3D and 5D translocations had Sr31, Lr26 and Yr9 but not Sec-1. The 7D translocation lacked Sec-1, Lr26 and Yr9, but the presence of Sr31 in this translocation was not determined. This showed that the translocation points fell into three regions of the 1R satellite, namely, proximal to Sec-1, between Sec-1 and the rust resistance loci, and distal to the rust resistance loci. Thus, the 3C gametocidal system was demonstrated to be effective in transferring small rye chromosome segments. This revised version was published online in July 2006 with corrections to the Cover Date.     

Normal haematology of free-living green sea turtles ( Chelonia mydas ) from the United Arab Emirates

A comprehensive health assessment was conducted in May 1997 on free-living green sea turtles (Chelonia mydas) occurring off the coast of Ras Al-Khaima, United Arab Emirates. As an integral part of this assessment, blood samples were obtained from a total of 72 clinically normal individuals, including 25 adult females, 20 adult males, 14 subadults and 13 juvenile turtles to establish normal haematology reference values for free-living green sea turtles.     

Characterization of a Plasmodium chabaudi gene encoding a protein with glutamate-rich tandem repeats

Several highly antigenic proteins containing tandem repeats rich in glutamic acid residues have been described in Plasmodium falciparum. However, relatively little information is available about analogous genes in rodent parasites. This report describes a 4.2-kb genomic DNA fragment from P. chabaudi with a deduced amino acid sequence that is predominantly glutamate-rich tandem repeats. Several different monoclonal antibodies raised against a 93-kDa P. chabaudi protein, which does not correspond to the cloned DNA fragment, recognize a recombinant protein expressed from the 4.2-kb DNA fragment. The only sequence similarities between these two genes are tandem repeats with a predominance of glutamate pairs followed by a hydrophobic residue. This repetitious-sequence motif may be the basis for the observed cross-reactivity. A similar motif has been demonstrated to be the basis for antibody cross-reactivity between glutamate-rich proteins of P. falciparum. The expression of multiple glutamate-rich proteins with cross-reacting epitopes may be a general phenomenon in Plasmodium species. Received: 3 March 1998 / Accepted: 7 July 1998     

Effect of temperature on the development of free-living stages of <Emphasis Type="Italic">Strongyloides ratti</Emphasis>

In the genus Strongyloides, larval development external to the host is known to be markedly affected by a variety of environmental factors. This investigation focuses on the effect of temperature on Strongyloides ratti. Low temperature (15°C) was shown to favor direct development, producing infective larvae, while high temperature (25°C) favored indirect development, producing free-living females and males. Different courses of development were brought about by either a 16-h temperature stimulus at 15°C or a 6-h temperature stimulus at 25°C. Moreover, eggs were not susceptible to the cold-temperature stimulus of 15°C, while newly hatched larvae were. The results indicate that the developmental course of S. ratti larvae external to the host is determined at a relatively early stage before the first molt.