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 <5% and >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.

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.

Study of trace elements in stranded green turtles (Chelonia mydas), hawksbill turtles (Eretmochelys imbricata), and olive ridley turtles (Lepidochelys olivacea) in Gulf of Thailand and Andaman Sea.

The purpose of this study was to survey and compare the amounts of elements in the serum of stranded sea turtles from the Gulf of Thailand and the Andaman Sea. The sea turtles from the Gulf of Thailand had Ca, Mg, P, S, Se, and Si concentrations significantly higher than those in sea turtles from the Andaman Sea. The Ni and Pb concentrations of sea turtles from the Gulf of Thailand was higher, but not significantly so, than in sea turtles from the Andaman Sea. Rb was detected only in sea turtles from the Gulf of Thailand. This may have been related to the industrial activities in Eastern Thailand. The concentration of Br in the sea turtles from the Andaman Sea were significantly higher than those in sea turtles from the Gulf of Thailand. The higher serum concentration of Cu in hawksbill (H) and olive ridley turtles (O) than in green turtles may be due to hemocyanin, as an important component in the blood of crustaceans. The higher Fe concentration in the serum from green turtles than for H and O may be due to chlorophyll, which is an important component of chloroplasts in eel grass. Co was not found in the serum of green turtles but was found in the serum of H and O. The monitoring of important elements in sea turtles may be used as a tool to assess the levels of pollution in marine ecosystems.

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.

Association of bacterial isolates and antimicrobial susceptibility between prostatic fluid and urine samples in canine prostatitis with concurrent cystitis.

Most old, intact male dogs usually have prostate disorders, especially benign prostatic hypertrophy and prostatitis with or without abscesses, and concurrent cystitis. The successful treatment of dogs with prostatitis concurrent with cystitis has relied on choosing an appropriate antimicrobial drug based on a bacterial culture and drug sensitivity testing. The objective of the study was to compare the prevalence of bacterial species and results of drug susceptibility testing of bacteria that were isolated from the prostatic fluids and urine samples that were collected from dogs with both prostatitis and cystitis. One hundred and sixty intact male dogs, who presented with both diseases, were recruited for the study. The disease diagnoses were based on clinical history notes, physical examinations, abdominal ultrasonography, prostatic fluid cytology, urinalysis and bacterial cultures from both prostatic fluid and urine samples. The bacterial culture results demonstrated that the major species that were detected in either the prostatic fluid or urine samples were Staphylococcus spp., Escherichia coli, Pseudomonas spp., Streptococcus spp., Proteus mirabilis and Klebsiella pneumoniae. Staphylococcus spp. (26.5 %, 43/162) and Escherichia coli (26.1 %, 12/46) were the most prevalent species from the prostatic fluid and urine samples, respectively. Statistical tests revealed that there were no significantly different prevalence levels among the isolated bacteria between the prostatic fluid and urine samples. Imipenem and gentamicin were the most potent antimicrobial drugs tested against the bacterial isolates in the present study. However, the administration of imipenem to treat prostatitis and cystitis in dogs was of concern. Interestingly, there were no significant differences in the antimicrobial drug susceptibility trends between the prostatic fluid and urine samples. Based on these results, a urine sample might be considered as an optional sample for bacterial cultures and antimicrobial drug susceptibility testing when it is not possible to collect a prostatic fluid sample.

Expression of recombinant 35 kDa fragment of VP2 protein of canine parvovirus using Escherichia coli expression system.

BACKGROUND AND AIM: Canine parvovirus (CPV) is one of the most common viral infections in dogs, causing acute hemorrhagic gastroenteritis and high mortality. Vaccination effectively prevents CPV infection. However, the currently available CPV vaccines have concerns such as maternal immunity interference, shedding of virus vaccine, and false-positive result based on polymerase chain reaction after vaccination. A subunit vaccine can overcome these problems. This study aimed to express the recombinant 35 kDa fragment of the VP2 protein (consisting of epitopes 1-7) and the recombinant full-length VP2 protein (consisting of epitopes 1-10) and to study the ability of these two recombinant proteins to react with rabbit anti-CPV polyclonal antibodies. MATERIALS AND METHODS: The full length and 35 kDa fragment of VP2 gene of CPV were cloned into the pBAD202 Directional TOPO™ expression vector and expressed in E. coli. The recombinant full-length and the recombinant 35 kDa fragment proteins of VP2 were analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting. RESULTS: The recombinant full-length and the recombinant 35 kDa fragment VP2 genes were successfully cloned and expressed. The optimum concentrations of arabinose and induction time for the recombinant full-length and the recombinant 35 kDa fragment VP2 proteins were 0.2% for 6 h and 0.02% for 6 h, respectively. The recombinant full-length and the recombinant 35 kDa fragment VP2 protein molecular weights were approximately 81 and 51 kDa, respectively. The recombinant full-length and the recombinant 35 kDa fragment VP2 proteins specifically interacted with rabbit anti-CPV polyclonal antibodies. CONCLUSION: These results suggest that the recombinant 35 kDa fragment and the recombinant full-length VP2 proteins may be useful in developing a CPV diagnostic test or vaccine.

Molecular studies on oestrogen α and progesterone receptors and histomorphometric analysis of canine uteri following aglepristone treatment.

Aglepristone, a competitive progesterone antagonist, is successfully used in various progesterone-dependent conditions. This study investigated uterine histomorphometric analysis, and expressions of the oestrogen α receptor (ERα) and progesterone receptor (PR) in uteri of bitches following the single dose of aglepristone treatment. Twelve client-owned healthy diestrous bitches were used in the study. The single dose of aglepristone (Alizine® , 10 mg/kg) was injected subcutaneously 5 days before ovariohysterectomy in the treatment group (n = 6); bitches without treatment served as a control group (n = 6). Uteri were collected for histomorphometric analysis, ERα and PR gene, and protein expressions studies. The mRNA expressions of ERα and PR were determined by RT-qPCR. Immunohistochemical analysis was used to evaluate the ERα and PR protein expressions using an H-score in five parts of the uterus. The results demonstrated glandular epithelium height significantly decreased (p < .05) and ERα mRNA increased (p < .01) in treated dogs. Of the treated bitches, lower expression levels of ERα were observed in the luminal epithelium, crypt and glandular epithelium, with higher expression in the endometrial stroma and myometrium (p < .05); however, PR expression decreased in the luminal epithelium, crypt and glandular epithelium (p < .01). In conclusion, reduction of the uterine glandular epithelium and ERα mRNA upregulation together with changes in ERα and PR expressions were observed in the treated bitches. However, changes in uterine ERα and PR expressions in the treated bitches depended on tissue layers. The treatment had no effect on serum oestradiol and progesterone levels.

Expression of the recombinant C-terminal of the S1 domain and N-terminal of the S2 domain of the spike protein of porcine epidemic diarrhea virus.

BACKGROUND AND AIM: Porcine epidemic diarrhea virus (PEDV) causes severe diarrhea in suckling piglets, leading to severe economic losses in the swine industry. Commercial vaccines have limited effectiveness against different genogroups of PEDV and the shedding of virus. The C-terminal of the S1 domain and the N-terminal of the S2 domain (S1-2) protein of the spike (S) protein have four neutralizing epitopes. However, research on the expression of the S1-2 segment of the S gene has been limited. In this study, we expressed a recombinant S1-2 protein of the S protein of the PEDV Thai isolate and characterized the immunological properties of the recombinant S1-2 protein. MATERIALS AND METHODS: The S1-2 segment of the S gene of the PEDV Thai isolate (G2b) was amplified, cloned into the pBAD202/D-TOPO® vector (Invitrogen, Carlsbad, CA, USA), and expressed in Escherichia coli. The optimum concentration of arabinose and the optimum induction time for the expression of the recombinant S1-2 protein were determined using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The immunogenic reactivity of the recombinant S1-2 protein was determined using Western blot analysis with rabbit polyclonal antibodies against the SM98 strain of PEDV (G1a). RESULTS: The recombinant S1-2 segment of the S gene of the PEDV Thai isolate protein was cloned and the recombinant S1-2 protein was successfully expressed. The optimum concentration of arabinose and the optimum induction time for the induction of the recombinant S1-2 protein were 0.2% and 8 h, respectively. The recombinant S1-2 protein reacted specifically with both rabbit anti-histidine polyclonal antibodies and rabbit anti-PEDV polyclonal antibodies. CONCLUSION: The recombinant S1-2 protein reacted with rabbit anti-PEDV polyclonal antibodies induced by the different PEDV genogroup. Therefore, the recombinant S1-2 protein may be a useful tool for the development of a diagnostic test for PEDV or for a vaccine against PEDV.

Feline panleukopenia virus as the cause of diarrhea in a banded linsang (Prionodon linsang) in Thailand.

A banded linsang (Prionodon linsang) presented at our hospital with clinical signs of acute diarrhea. Fecal samples were positive for canine parvovirus (CPV) as determined by polymerase chain reaction with primers specific for both CPV and feline panleukopenia virus (FPV). The full-length VP2 was cloned, sequenced, and compared with sequences of FPV and CPV strains reported in GenBank. The amino acids that determined the host range were similar to those of FPV. Moreover, amino acid analysis of VP2 revealed over 98% homology to FPV. The FPV isolate was closely related with FPV isolates from Japan, South Korea, and China. To the best of our knowledge, this is the first study to report that banded linsang can be infected with FPV.

Sequence analysis of VP2 gene of canine parvovirus isolates in Thailand.

Canine parvovirus (CPV) causes a very severe enteric disease especially in puppies. Twenty-six isolates of CPV were obtained from dogs at the Animal Hospital, Kasetsart University, Thailand. Whole VP2 gene of 26 isolates was amplified using polymerase chain reaction (PCR) and its sequences were analyzed. Nineteen out of 26 isolates were characterized as CPV type 2a variants and the rest of the isolates were characterized as CPV type 2b. These results indicated that both types are currently prevalent field CPV circulating in Thailand and type 2a is the predominant genotype. Neither CPV type 2 nor type 2c was observed in this study.

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

BACKGROUND: 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. METHODS: 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 (1000x). Normal sperm head morphometry characteristics were measured by Computer-Assisted Semen Analysis (Hamilton Thorne, USA.) after applying the Harris' haematoxylin staining technique. RESULTS: 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. CONCLUSION: 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.

Finasteride-induced prostatic involution by apoptosis in dogs with benign prostatic hypertrophy.

OBJECTIVE: To determine the effect of finasteride on programmed cell death (apoptosis) of prostatic cells during prostatic involution in dogs with benign prostatic hypertrophy (BPH). ANIMALS: 9 dogs with BPH. PROCEDURE: Dogs were randomly assigned to treatment or control groups. Treatment dogs (n = 5) were administered finasteride (0.1 to 0.5 mg/kg, PO, q 24 h) for 16 weeks, whereas the 4 control dogs were administered an inert compound. Prostatic cells from the prostatic fluid portion of the ejaculate of treatment and control dogs were obtained before and 1, 2, 3, 4, 8, and 16 weeks after initiation of treatment. Cells were concentrated by use of centrifugation. Prostatic cells were examined for indications of apoptosis by use of a terminal deoxyribonucleotidyl transferase-mediated deoxyuracil triphosphate nick-end labeling technique. After receiving the inert compound for 16 weeks, the 4 control dogs were administered finasteride for 16 weeks, and evaluations were repeated. RESULTS: Percentage of apoptotic prostatic cells in ejaculated prostatic fluid of treatment dogs increased significantly (from 9% before treatment to 33, 31, 26, and 27% after 1, 2, 3, and 8 weeks of treatment, respectively). There was no significant change in percentage of apoptotic prostatic cells in the ejaculated prostatic fluid of control dogs. CONCLUSIONS AND CLINICAL RELEVANCE: Finasteride-induced prostatic involution appears to be via apoptosis in dogs with BPH. Finasteride treatment of dogs with BPH causes prostatic involution by apoptosis rather than necrosis.