Genetic structure and diversity of green turtle (Chelonia mydas) in the Gulf of Thailand

Background and Aim: The International Union for the Conservation of Nature and Natural Resources lists the green turtle as endangered. Green turtle nesting behavior in the Gulf of Thailand has decreased to <50% of the 1995 level. The population structure of green turtles in the Gulf of Thailand has not yet been studied. This study aimed to characterize the genetic diversity of green turtles in the Gulf of Thailand based on comparisons of mitochondrial DNA (mtDNA) control region with sequences of Indo-Pacific management units (MUs) and rookeries, to investigate population structures, and to explore phylogeographic relationships. Materials and Methods: Blood samples (1 mL each) from 91 stranded green turtles were collected from four parts of the Gulf of Thailand (eastern, upper, central, and lower). The control mtDNA region was amplified by polymerase chain reaction using LCM15382 and H950 primer. The obtained 384-bp or 770-bp sequences were analyzed for haplotype, clade, and haplotype and nucleotide diversities and were used to construct a phylogenetic tree and haplotype network diagram, respectively. In addition, we analyzed genetic differentiation within and among populations of green turtles in the Gulf of Thailand and between green turtles in the Gulf of Thailand and other Indo- Pacific MUs and rookeries. Results: In total, 12 (based on 384 bp) or 13 (based on 770 bp) haplotypes and two clades (clades VII and VIII) were identified, with nine or 10 haplotypes belonging to clade VIII and three haplotypes belonging to clade VII. Of the new haplotypes, four or five were identified and classified as clade VII (two haplotypes, for both fragment lengths) and clade VIII (two or three haplotypes, for 384 bp or 770 bp fragments, respectively). The overall haplotype and nucleotide diversity of green turtles in the Gulf of Thailand were high (0.755 ± 0.039 and 0.01146 ± 0.00248, respectively). Based on the analysis of molecular variance, green turtles in the Gulf of Thailand could be divided into two subpopulations (UC-Eastern Gulf of Thailand [UC-EGT] and lower Gulf of Thailand [LGT]). Comparisons with other MUs and rookeries in the Indo-Pacific showed that UC-EGT was not genetically different from the Peninsular Malaysia and Eastern Taiwan (Lanyu) MUs and the Terrangganu and Mersing rookeries, and LGT were not genetically different from Peninsular Malaysia, Sipadan, Brunei Bay, Eastern Taiwan (Lanyu), Scott Reef and Browse Island, and Gulf of Carpentaria MUs and the Perak, Perhentain Island, Redang, Pahang, and Vietnam rookeries. Conclusion: To the best of our knowledge, this is the first report to identify the haplotypes and clades of green turtles in the Gulf of Thailand and to show that the populations in the Gulf of Thailand not only present high genetic diversity but also have haplotypic endemism. Longer mtDNA fragments (770 bp) increased the resolution of the stock structure. Clade VII is a unique clade not only for Japan but also for Thailand and Malaysia, and CmP82 is a unique haplotype for both the Gulf of Thailand and Malaysia. Conservation and management of these populations are important to preserve the genetic diversity, biological diversity, and evolutionary potential of green turtles in the Gulf of Thailand

Morphology and head morphometric characters of sperm in Thai native crossbred stallions

<p>Abstract</p> <p>Background</p> <p>One of the semen quality parameters use to determine fertility is the percentage of sperm that express normal morphology. Sperm head morphometry is also correlated with fertility. The objectives of this study were 1) to investigate the sperm morphology and normal sperm head morphometry of Thai native crossbred stallions, and 2) to compare our results with the characteristics of proven fertile sperm from purebred stallions.</p> <p>Methods</p> <p>Semen samples were collected monthly from nine stallions, of which five were Thai native crossbred (T) and four were purebred of proven fertility (F: F1 was a Standard-bred; F2 was a Warm-blood; F3 and F4 were Thoroughbreds). All the animals were aged between 5 and 12 years. Sperm morphological examination was performed using formaldehyde-fixed samples under phase-contrast microscopy (1000×). Normal sperm head morphometry characteristics were measured by Computer-Assisted Semen Analysis (Hamilton Thorne, USA.) after applying the Harris' haematoxylin staining technique.</p> <p>Results</p> <p>The percentages of morphologically normal and abnormal sperm varied among individual stallions in both the T and F groups. The mean percentage of morphologically normal sperm was not significantly different (P > 0.05) between T and F stallions (mean ± SE, 49.7 ± 1.3 and 48.1 ± 2.8, respectively). A comparison between the T and F sperm heads revealed that all the dimensional parameters were significantly different (P < 0.05). The coefficients of within-animal variation (CVs) ranged from 2.6 (shape factor 1) to 7.5 (elongation) and 2.9 (shape factor 1) to 8.1 (elongation) in T and F, respectively. In the case of the T group, those sperm head parameters that featured a low within-animal CV and a high between-animal CV were perimeter (2.9, 19.1), shape factor 1 (2.6, 25.8) and shape factor 3 (3.8, 32.0). In the case of the F group, only shape factor 1 (2.9, 26.1) featured such characteristics.</p> <p>Conclusion</p> <p>We found variability in the percentage of morphologically normal and abnormal sperm, as well as in sperm head dimensions among Thai native crossbred stallions, and these results were similar to those of purebred stallions. Our findings demonstrate that the heads of the T sperm specimens were larger and rounder than that of the F sperm. Perimeter, shape factor 1 and shape factor 3 could be used as parameters for the identification of individual T stallions based on a sperm sample.</p

First report on clinical aspects, blood profiles, bacterial isolation, antimicrobial susceptibility, and histopathology in canine pyometra in Thailand

Background and Aim: Canine pyometra, either the closed (closed pyometra [CP]) or open (open pyometra [OP]) cervix type, is a frequent uterine disease in intact old age bitches. Therefore, early diagnosis and appropriate medical and surgical treatments are crucial to avoid the life-threatening condition in these bitches. This study aimed to investigate clinical alterations, blood parameters, causative bacteria, antimicrobial susceptibility, and uterine histopathology obtained during aseptic surgical treatment on bitches with pyometra. Materials and Methods: Sixty bitches of various breeds and ages with presumptive pyometra diagnoses were included in the study. The diagnoses were based on history, clinical examination, blood parameters, radiography, and ultrasonography. All pyometra bitches were ovariohysterectomized as an emergency surgical treatment. In addition, uterine content and tissues were submitted for bacterial isolation, antimicrobial susceptibility, and uterine histopathological analysis. Results: Except for abdominal CP distention, no specific clinical signs were linked to the pyometra type. The mean values of total white blood cell count (WBC) and plasma protein were predominantly raised in pyometra bitches regarding hematological parameters. Leukocytosis was found in both types; however, the WBC in CP was markedly higher than in OP. The mean value of blood urea nitrogen increased in the CP group. Klebsiella pneumoniae and Escherichia coli were the most frequent causative bacteria isolated in CP and OP, respectively. All isolated bacteria were 100% susceptible to imipenem, meropenem, and carbapenem. Marbofloxacin was the second most effective drug against isolated bacteria from both groups. Uncomplicated cystic endometrial hyperplasia (CEH) was not presented in the CP group. CEH and chronic endometritis (type IV), the most severe uterine histopathological changes, were discovered in the CP and OP. Conclusion: The CP and OP groups presented leukocytosis, increased plasma protein, and CEH and chronic endometritis. Depression, abdominal distention, and enlarged uterine size were the major characteristics of the CP group. Furthermore, abdominal distension is presented in other abnormalities in clinical practices, providing a differential diagnosis. Drugs in the carbapenem group were the most effective against isolated bacteria; however, they are not routinely used due to bacterial resistance concerns. Thus, marbofloxacin was recommended as an alternative medical treatment because it is convenient to manage by both oral and injection routes

Proliferation and apoptosis studies of interplacental areas after aglepristone treatment for planned cesarean section in pregnant bitches

Background and Aim: Progesterone (P4) is the main hormone for pregnancy maintenance, occurring approximately 62–64 days after ovulation in bitches. Progesterone acts by binding to specific receptors. Aglepristone is a progesterone receptor (PR) antagonist with a higher affinity for PR binding. There are no published studies on cell proliferation and apoptosis in the canine uterus at the time of parturition. Therefore, this study aimed to determine the local effects of aglepristone on cell proliferation and apoptosis of interplacental uterine tissue during planned cesarean section (C-section) in bitches. Materials and Methods: In this study, 13 client-owned French bulldogs were examined. Bitches were divided into treatment (n = 8) and control (n = 5) groups. Ovulation timing was predicted based on the serum P4 level on 62–64 days post-ovulation for parturition. Serum P4 levels were measured before (on 60-day post-ovulation) and on C-section day (on 61-day post-ovulation). Aglepristone (Alizine®), 15 mg/kg subcutaneously (SC), was administered on 60 days post-ovulation in the treatment group. A C-section was planned 20–24 h later, and interplacental uterine areas were collected from both groups during the C-section. Immunohistochemistry based on Ki-67 and TUNEL assay was used to evaluate cell proliferation and apoptosis in four different interplacental uterine tissue layers (epithelium, stroma, glandular epithelium, and myometrium). Data are reported as mean ± standard deviation. Kruskal–Wallis test was used for comparisons of more than two independent groups. P value of 0.05 was considered statistically significant. Results: One bitch in the treatment group was excluded due to emergency C-section 8 h after aglepristone administration. Serum P4 levels (ng/mL) at 20–24 h before and at C-section were 6.09 ± 2.72 and 4.32 ± 2.2 in the treatment group (n = 7) and 5.45 ± 1.28 and 3.67 ± 1.89 in the control group (n = 5), respectively. Proliferation (PI) and apoptotic (AI) indices were 45%, respectively, in both the treatment (n = 5) and control (n = 3) groups. PI and AI were detected at interplacental areas. Conclusion: There were no significant differences in serum P4 levels or PI and AI indices between the groups. The PI <5% and AI was higher than 45% in both groups. Aglepristone did not have a direct effect on the serum P4 levels in both groups. These results correlated with the natural physiology of parturition preparation. Aglepristone 15 mg/kg SC injected 20–24 h before parturition had no effect on the P4 level, nor were any harmful effects observed for a planned C-section in pregnant bitches

Interplay between Interferon-Mediated Innate Immunity and Porcine Reproductive and Respiratory Syndrome Virus

Innate immunity is the first line of defense against viral infection, and in turn, viruses have evolved to evade host immune surveillance. As a result, viruses may persist in host and develop chronic infections. Type I interferons (IFN-α/β) are among the most potent antiviral cytokines triggered by viral infections. Porcine reproductive and respiratory syndrome (PRRS) is a disease of pigs that is characterized by negligible induction of type I IFNs and viral persistence for an extended period. For IFN production, RIG-I/MDA5 and JAK-STAT pathways are two major signaling pathways, and recent studies indicate that PRRS virus is armed to modulate type I IFN responses during infection. This review describes the viral strategies for modulation of type I IFN responses. At least three non–structural proteins (Nsp1, Nsp2, and Nsp11) and a structural protein (N nucleocapsid protein) have been identified and characterized to play roles in the IFN suppression and NF-κB pathways. Nsp’s are early proteins while N is a late protein, suggesting that additional signaling pathways may be involved in addition to the IFN pathway. The understanding of molecular bases for virus-mediated modulation of host innate immune signaling will help us design new generation vaccines and control PRRS

Genome-wide transcriptional response of primary alveolar macrophages following infection with porcine reproductive and respiratory syndrome virus

Porcine reproductive and respiratory syndrome is a major cause of economic loss for the swine industry worldwide. Porcine reproductive and respiratory syndrome virus (PRRSV) triggers weak and atypical innate immune responses, but key genes and mechanisms by which the virus interferes with the host innate immunity have not yet been elucidated. In this study, genes that control the response of the main target of PRRSV, porcine alveolar macrophages (PAMs), were profiled in vitro with a time-course experiment spanning the first round of virus replication. PAMs were obtained from six piglets and challenged with the Lelystad PRRSV strain, and gene expression was investigated using Affymetrix microarrays and real-time PCR. Of the 1409 differentially expressed transcripts identified by analysis of variance, two, five, 25, 16 and 100 differed from controls by a minimum of 1.5-fold at 1, 3, 6, 9 and 12 h post-infection (p.i.), respectively. A PRRSV infection effect was detectable between 3 and 6 h p.i., and was characterized by a consistent downregulation of gene expression, followed by the start of the host innate immune response at 9 h p.i. The expression of beta interferon 1 (IFN-β), but not of IFN-α, was strongly upregulated, whilst few genes commonly expressed in response to viral infections and/or induced by interferons were found to be differentially expressed. A predominance of anti-apoptotic transcripts (e.g. interleukin-10), a shift towards a T-helper cell type 2 response and a weak upregulation of tumour necrosis factor-α expression were observed within 12 h p.i., reinforcing the hypotheses that PRRSV has developed sophisticated mechanisms to escape the host defence

A Rapid and Highly Sensitive Method of Non Radioactive Colorimetric In Situ Hybridization for the Detection of mRNA on Tissue Sections

Background: Non Radioactive colorimetric In Situ Hybridization (NoRISH) with hapten labeled probes has been widely used for the study of gene expression in development, homeostasis and disease. However, improvement in the sensitivity of the method is still needed to allow for the analysis of genes expressed at low levels. Methodology/Principal Findings: A stable, non-toxic, zinc-based fixative was tested in NoRISH experiments on sections of mouse embryos using four probes (Lhx6, Lhx7, ncapg and ret) that have different spatial patterns and expression levels. We showed that Z7 can successfully replace paraformaldehyde used so far for tissue fixation in NoRISH; the morphology of the cryosections of Z7-fixed tissues was excellent, and the fixation time required for tissues sized 1 cm was 1 hr instead of 24 hr for paraformaldehyde. The hybridization signal on the sections of the Z7-treated embryos always appeared earlier than that of the PFA-fixed embryos. In addition, a 50–60 % shorter detection time was observed in specimen of Z7-treated embryos, reducing significantly the time required to complete the method. Finally and most importantly, the strength of the hybridization signal on the sections of the Z7-treated embryos always compared favorably to that of the sections of PFAfixed embryos; these data demonstrate a significant improvement of the sensitivity the method that allows for the analysis of mRNAs that are barely or not detected by the standard colorimetric NoRISH method. Conclusions/Significance: Our NoRISH method provides excellent preservation of tissue morphology, is rapid, highl

Coronavirus Gene 7 Counteracts Host Defenses and Modulates Virus Virulence

Transmissible gastroenteritis virus (TGEV) genome contains three accessory genes: 3a, 3b and 7. Gene 7 is only present in members of coronavirus genus a1, and encodes a hydrophobic protein of 78 aa. To study gene 7 function, a recombinant TGEV virus lacking gene 7 was engineered (rTGEV-Δ7). Both the mutant and the parental (rTGEV-wt) viruses showed the same growth and viral RNA accumulation kinetics in tissue cultures. Nevertheless, cells infected with rTGEV-Δ7 virus showed an increased cytopathic effect caused by an enhanced apoptosis mediated by caspase activation. Macromolecular synthesis analysis showed that rTGEV-Δ7 virus infection led to host translational shut-off and increased cellular RNA degradation compared with rTGEV-wt infection. An increase of eukaryotic translation initiation factor 2 (eIF2α) phosphorylation and an enhanced nuclease, most likely RNase L, activity were observed in rTGEV-Δ7 virus infected cells. These results suggested that the removal of gene 7 promoted an intensified dsRNA-activated host antiviral response. In protein 7 a conserved sequence motif that potentially mediates binding to protein phosphatase 1 catalytic subunit (PP1c), a key regulator of the cell antiviral defenses, was identified. We postulated that TGEV protein 7 may counteract host antiviral response by its association with PP1c. In fact, pull-down assays demonstrated the interaction between TGEV protein 7, but not a protein 7 mutant lacking PP1c binding motif, with PP1. Moreover, the interaction between protein 7 and PP1 was required, during the infection, for eIF2α dephosphorylation and inhibition of cell RNA degradation. Inoculation of newborn piglets with rTGEV-Δ7 and rTGEV-wt viruses showed that rTGEV-Δ7 virus presented accelerated growth kinetics and pathology compared with the parental virus. Overall, the results indicated that gene 7 counteracted host cell defenses, and modified TGEV persistence increasing TGEV survival. Therefore, the acquisition of gene 7 by the TGEV genome most likely has provided a selective advantage to the virus

Development of molecular techniques for the detection and pathogenesis study of swine corona-and corona-like virus

In situ hybridization (ISH) technique was first developed to detect and differentiate transmissible gastroenteritis virus (TGEV) and porcine respiratory coronavirus (PRCV) in cell culture and tissue sections using 35S-labeled RNA probes. RNA probe generated from plasmid PSP.FP2 detected both TGEV and PRCV, whereas PSP.FP1 probe detected only TGEV. The TGEV RNA was detected mainly within the enterocytes at the tips of villi and within a few crypt epithelial cells. The PRCV RNA was detected mainly in the bronchiolar epithelial cells and in lesser amount in type I and type II pneumocytes, alveolar macrophages and bronchial epithelial cells. Since the ISH technique using a radiolabeled probe is time consuming and not user friendly, the nonisotopic ISH technique using a fluorescein-labeled RNA probe was developed.;A rapid ISH technique using radiolabeled and fluorescein-labeled probes was able to decrease hybridization time from 20 hours to 2 hours without compromising the intensity of the signal and tissue morphology. By the rapid nonisotopic ISH technique, the entire procedure could be performed within about 7-8 hours. We demonstrated TGEV induced apoptosis in swine testes cell cultures by gel electrophoresis, electron microscopy, and terminal deoxytransferase digoxigenin-dUTP nick end labeling (TUNEL) technique. By electron microscopy, we showed that infected-ST cells from TGEV-inoculated wells were undergoing cell lysis, however, uninfected-ST cells were undergoing apoptosis. Double labeling technique also demonstrated that TGEV positive cells were negative for apoptosis and apoptotic cells were negative for TGEV RNA. Our results indicated that TGEV induced apoptosis in uninfected bystander cells, thus amplifying the cytopathic effect of TGEV.;Porcine reproductive and respiratory syndrome virus (PRRSV) is an economically emerging virus in swine. We demonstrated that PRRSV induced apoptosis both in vitro and in vivo and apoptotic cells were uninfected bystander cells. In the lungs of PRRSV-infected pigs, the apoptotic cells were predominantly alveolar macrophages, lymphocytes, pulmonary intravascular macrophages, and type I and type II pneumocytes. In the lymph nodes of PRRSV-infected pigs, the apoptotic cells were predominantly lymphocytes and macrophages. A large number of macrophages and lymphocytes undergoing apoptosis might be the reason that PRRSV-infected pigs are susceptible to secondary infection.</p

Extender for Sperm Dilution in Olive Ridley Turtle (Lepidochelys olivacea) and Hawksbill Turtle (Eretmochelys imbricata) Semen

March 5-6, 2009, Bangkok, ThailandThe objective of the study was to find an extender to dilute and preserve sea turtle semen. Six adult olive ridley turtles (Lepidochelys olivacea), and 4 adult hawksbill turtles (Eretmochelys imbricata) from Phuket Marine Biological Center and Eastern Marine and Coastal Resources Research Center in Thailand had semen collected using electroejaculator. The study was repeated twice in 2007 and 2008. After collection, each semen sample was divided and preserved in 8 different extenders, which were 1) refrigeration medium test yolk buffer, 2) Tyrode medium supplemented with albumin, lactate and pyruvate, 3) Beltsville poultry semen extender, 4) 3% Sodium citrate buffer, 5) Phosphatebuffered solution, 6) EEL extender, 7) 1% bovine serum albumin, and 8) HAM F-10 and kept at 4℃ and evaluated for viability (motile sperm) at 0, 0.25, 0.5, 1, 3, 6, 12, 24 and 48 hours. The results found that for 28% olive ridley turtles and 25% hawksbill turtles that their spermatozoa diluted in extender 1, and for 14% of both sea turtles that their spermatozoa diluted in extender 2 could survive for 24 hours. However, the motility from both sea turtles semen in both extenders was decreased by 50-80 %. Most sperm died after being diluted in the last 6 extenders. By conclusion, extender 1 and 2 were suitable extenders for sea turtle semen viability, however, adding other ingredients should be considered to enhance in viability