Antimicrobial resistance profiles in bacterial species isolated from fecal samples of free-ranging long-tailed macaques (Macaca fascicularis) living in Lopburi Old Town, Thailand.

BACKGROUND AND AIM: At present, increasing in long-tailed macaques (Macaca fascicularis) population in Lopburi old town caused several problems in its community, in particular with sanitation problem. The present study aimed to explore species distribution and antimicrobial resistance patterns in bacteria isolated from feces of the free-ranging long-tailed macaques (Macaca fascicularis) in Lopburi Old Town, Thailand. MATERIALS AND METHODS: Fresh fecal samples were collected from October 2018 to July 2019 from seven troops of macaques. Bacterial colonies were identified based on Gram stain and standard biochemical techniques. Sensitivity toward eight different antibiotics, including amoxicillin, amoxicillin-clavulanate, cephalexin, clindamycin, doxycycline, enrofloxacin, erythromycin, and gentamicin, was analyzed using the disk diffusion method. RESULTS: A total of 1050 fecal samples were collected. Five unique bacterial species were identified, including Escherichia coli, Enterobacter spp., Proteus spp., Salmonella Group B, and Citrobacter spp. in 100%, 25.71%, 18%, 1.71%, and 0.57% of the fecal specimens, respectively. Among 70 distinct isolates of E. coli, 63 (93%) were resistant to multiple drugs, including amoxicillin, cephalexin, clindamycin, and erythromycin; one isolate (6%) was resistant to clindamycin only. Furthermore, 17 isolates (94%) of Salmonella Group B were resistant to both clindamycin and erythromycin. Five of the six Citrobacter spp. isolates (83%) were also multidrug-resistant (to cephalexin, clindamycin, and erythromycin); the one remaining Citrobacter spp. isolate (6%) was resistant to both clindamycin and erythromycin. However, a high percentage of E. coli, Salmonella Group B and Citrobacter spp. remained susceptible to amoxicillin-clavulanate, enrofloxacin, and doxycycline. CONCLUSION: Our findings provide the basic information for the selection of empirical therapy and for the evaluation of the scale of antibiotic resistance associated with macaques living in Lopburi Old Town.

Gonadosomatic Index, Oocyte Development and Fecundity of the Snakehead Fish (Channa striata) in Natural River of Mae La, Singburi Province, Thailand.

BACKGROUND AND OBJECTIVE: Reproductive cycle of snakehead fish (Channa striata) is necessary to inform management plans of the natural fish. This work aimed to study the gonadosomatic index (GSI), oocyte development and fecundity of C. striata in the Mae La River, Singburi province, Thailand from November, 2017-October, 2018. MATERIALS AND METHODS: GSI was calculated from gonadal weight and body weight. Ovarian sections derived from standard histological methods were used to evaluate oocyte development, respectively. Fecundity was measured using the gravimetric method and the relationships of fecundity-total length, fecundity-body weight and fecundity-ovarian weight were evaluated. RESULTS: The highest average GSI values for females and males were found in July (6.15±0.20 and 0.14±0.12, respectively). Ovarian histology revealed 4 stages of ovarian development: (1) immature, (2) maturing, (3) mature and (4) spent. Stages 1, 2, 3 and 4 were observed for 11, 10, 6 and 2 months, respectively. The highest percentages of immature, mature and spent oocytes were found in October (93.33%), July (89.56%) and August (33.33%), respectively. Fecundity ranged from 4,160-46,890. Fecundity varied depending on total length, body weight and ovarian weight, with correlation coefficients (R) of 0.986, 0.960 and 0.989, respectively. CONCLUSION: Oocyte maturation period of C. striata occurs from April to August. The highest gonads development was in July. Fecundity exhibited linear relationships with body length, body weight and ovarian weight.

Borrelia sp. phylogenetically different from Lyme disease- and relapsing fever-related Borrelia spp. in Amblyomma varanense from Python reticulatus.

BACKGROUND: Species of the genus Borrelia are causative agents of Lyme disease and relapsing fever. Lyme disease is the most commonly reported vector-borne disease in the northern hemisphere. However, in some parts of the world Lyme borreliosis and relapsing fever may be caused by novel Borrelia genotypes. Herein, we report the presence of a Borrelia sp. in an Amblyomma varanense collected from Python reticulatus. METHODS: Ticks were collected from snakes, identified to species level and examined by PCR for the presence of Borrelia spp. flaB and 16S rRNA genes. Phylogenetic trees were constructed using the neighbour-joining method. RESULTS: Three A. varanense ticks collected from P. reticulatus were positive for a unique Borrelia sp., which was phylogenetically divergent from both Lyme disease- and relapsing fever-associated Borrelia spp. CONCLUSION: The results of this study suggest for the first time that there is a Borrelia sp. in A. varanense tick in the snake P. reticulatus that might be novel.

Effect of human beta-globin bacterial artificial chromosome transgenesis on embryo cryopreservation in mouse models.

The purpose of the present study was to investigate the efficiency of embryo cryopreservation for four transgenic (TG) thalassaemic mouse strains, which is a key element of the ongoing gene banking efforts for these high-value animals. Heterozygous TG embryos were produced by breeding four lines of TG males to wild-type (WT) females (C57BL/6J). Intact two-cell embryos were cryopreserved by vitrification in straws using 35% ethylene glycol. Survival rates of cryopreserved embryos ranged between 91.1% (102/112) and 93.6% (176/188) without significant differences between the lines. In contrast, the paternal line had a significant effect on the development of these embryos to the blastocyst stage, which ranged from 50.6% (92/182) to 77.5% (79/102). This effect was also noted following embryo transfers, with implantation rates varying from 17.3% (19/110) to 78.1% (35/45). The results demonstrate that the in vivo developmental potential is significantly influenced by TG line and reveal a specific line effect on cryosurvival. All bacterial artificial chromosome transgenic fetuses developed from vitrified-warmed embryos showed expression of the human beta-globin transgene. In conclusion, the present study shows a strong TG line effect on developmental competence following cryopreservation and the vitrification method was successful to bank the human beta-globin TG-expressing mouse strains.

Effects of vitrification procedures on subsequent development and ultrastructure of in vitro-matured swamp buffalo (Bubalus bubalis) oocytes.

The purpose of the present study was to investigate the effects of two vitrification procedures on developmental capacity and ultrastructural changes of matured swamp buffalo oocytes. In vitro-matured oocytes were vitrified by using 35 and 40% ethylene glycol as vitrification solution for solid surface vitrification (SSV) and in-straw vitrification (ISV), respectively. Survival rate of vitrified-warmed oocytes, evaluated on the basis of ooplasm homogeneity, oolemma integrity and zona pellucida intactness, as well as parthenogenetic blastocyst rates of vitrified-warmed oocytes were significantly higher with SSV (89.3 and 13.6%, respectively) than ISV (81.8 and 5.5%, respectively). However, they were still significantly lower than that of control oocytes (100 and 34.2%, respectively). For examining the ultrastructural changes, fresh, VS-exposed (ISV and SSV), and vitrified-warmed (ISV and SSV) oocytes were processed for transmission electron microscopy. In VS-exposed oocytes, reduction of microvilli abundance and damage of mitochondrial membrane were found only in the ISV group. In vitrified-warmed oocytes, however, it was clear that both methods of vitrification induced profound ultrastructural modifications to microvilli, mitochondria, oolemma and cortical granules as well as to the size and position of vesicles. Damaged mitochondria were, however, more abundant in ISV vitrified oocytes than in SSV vitrified oocytes, which correlated with the developmental data, showing the superiority of the SSV method. The present study demonstrated the feasibility of vitrification of in vitro-matured swamp buffalo oocytes.

Gene expression profiles and in vitro development following vitrification of pronuclear and 8-cell stage mouse embryos.

The analysis of differences in gene expression, responding to cryopreservation may explain some of the observed differences in further development of the preimplantation stage embryos. The aim of this study was to create a link, for the first time, between morphological/developmental observations and gene activity changes following cryopreservation of embryos. Efficiency of two vitrification methods, solid surface and in-straw vitrifications for pronuclear-stage mouse zygotes and 8-cell stage mouse embryos was compared based on morphological survival, blastocyst formation, and changes in embryonic gene expression. Both stages of embryos were vitrified by SSV using 35% ethylene glycol (EG) for vitrification solution (VS) and in-straw vitrification using 40% EG for VS. No significant differences were found between immediate survival rates of embryos vitrified by SSV and in-straw vitrification in both stages. Blastocyst rates were significantly higher with SSV and not significantly different from that of control. These results showed that SSV was more efficient than in-straw vitrification. Treatment with cytochalasin-b did not improve cryosurvival during SSV. The quantification of selected gene transcripts from single embryo (6 embryos/treatment group) were carried out by quantitative real-time RT-PCR. It was performed by adding 1/8 of each embryo cDNA to the PCR mix containing the specific primers to amplify housekeeping gene (beta-actin), heat shock protein gene (Hsp70), genes related to oxidative stress (MnSOD and CuSOD), cold stress (CirpB, Rbm3), and cell-cycle arrest (Trp53). We found upregulation of all six stress-related genes at 3 hr post-warming in pronuclear stage embryos. Expression of these genes showed much higher level (2-33-fold) in in-straw vitrification than in in vitro control embryos. In SSV-treated embryos we could detect only slight changes (0.3-2-fold). At 10 hr post-warming, all genes were downregulated in embryos vitrified by in-straw method. In SSV-treated group expression of Hsp70 showed slight increase and Trp53 showed decrease. In contrast to pronuclear stage, there was no clear pattern of gene expression changes after vitrification in 8-cell stage embryos. Several genes were upregulated both at 3 and 10 hr post-warming. Moreover, we found upregulation of beta-actin gene which we expected to use as a reference gene in in-straw treated embryos in both 3 and 10 hr post-warming, while in pronuclear stage embryos and in SSV treatment there was no effect on beta-actin expression level. There was no difference in gene expression between blastocysts developed from fresh or vitrified embryos. In conclusion, the real-time RT-PCR method from single embryo opened new opportunities for the understranding of molecular events following cryopreservation. The upregulation of stress-related genes at 3 hr post-warming in pronuclear stage embryos might have been an early indicator of reduced viability following in-straw vitrification in good correlation with the developmental data to blastocyst stage.