Antimicrobial resistance of avian pathogenic Escherichia coli isolated from broiler, layer, and breeder chickens.

Background and Aim: Antimicrobials are extensively used in poultry production for growth promotion as well as for the treatment and control of diseases, including avian pathogenic Escherichia coli (APEC). Poor selection, overuse, and misuse of antimicrobial agents may promote the emergence and dissemination of antimicrobial resistance (AMR) in APEC. This study aimed to assess antimicrobial susceptibility patterns and detect antibiotic resistance genes (ARGs) in APEC isolated from clinical cases of colibacillosis in commercial broiler, layer, and breeder chickens. Materials and Methods: A total of 487 APEC were isolated from 539 across 300 poultry farms in various regions of Nepal. Antimicrobial susceptibility patterns was determined using the Kirby-Bauer disk diffusion and broth microdilution methods. The index of AMR, such as multiple antibiotic resistance (MAR) index, resistance score (R-score), and multidrug resistance (MDR) profile, were determined. Polymerase chain reaction was employed to detect multiple ARGs and correlations between phenotypic and genotypic resistance were analyzed. Results: The prevalence of APEC was 91% (487/539). All of these isolates were found resistant to at least one antimicrobial agent, and 41.7% of the isolates were resistant against 8-9 different antimicrobials. The antibiogram of APEC isolates overall showed the highest resistance against ampicillin (99.4%), whereas the highest intermediate resistance was observed in enrofloxacin (92%). The MAR index and R-score showed significant differences between broiler and layers, as well as between broiler breeder and layers. The number of isolates that were resistant to at least one agent in three or more antimicrobial categories tested was 446 (91.6%) and were classified as MDR-positive isolates. The ARGs were identified in 439 (90.1%) APEC isolates, including the most detected mobilized colistin resistance (mcr1) which was detected in the highest (52.6%) isolates. Overall, resistance gene of beta-lactam (blaTEM), mcr1, resistance gene of sulphonamide (sul1) and resistance gene of tetracycline (tetB) (in broiler), were detected in significantly higher than other tested genes (p < 0.001). When examining the pair-wise correlations, a significant phenotype-phenotype correlation (p < 0.001) was observed between levofloxacin and ciprofloxacin, chloramphenicol and tetracycline with doxycycline. Similarly, a significant phenotype-genotype correlation (p < 0.001) was observed between chloramphenicol and the tetB, and colistin with blaTEM and resistance gene of quinolone (qnrA). Conclusion: In this study, the current state of APEC AMR in commercial chickens is revealed for the first time in Nepal. We deciphered the complex nature of AMR in APEC populations. This information of molecular surveillance is useful to combat AMR in APEC and to contribute to manage APEC associated diseases and develop policies and guidelines to enhance the commercial chicken production.

Reproduction and Fertility of Buffaloes in Nepal.

Water buffalo (Bubalus bubalis) in Nepal contributes 57% of the total milk and 36% of the total meat production in the country. The productive efficiency of Nepalese buffaloes is quite low, due mainly to subfertility and infertility. Delayed puberty and prolonged inter-calving intervals, attributed mainly by anestrus due to silent cyclicity and ovarian acyclicity, are the major forms of infertility in Nepalese buffaloes. Moreover, buffaloes in Nepal show a distinct seasonal breeding pattern, with July to December as the active breeding season, and with April to June and January to March as the low and transitional breeding seasons, respectively. Endoparasitic infection and poor nutritional status, which are more severe during the low season, are found to be the major factors causing anestrus and compromising its treatment response in buffaloes. Various hormonal protocols for timed artificial insemination (TAI) have been attempted, with a varying pregnancy outcome. Recently, an integrated technique including anthelmintic treatment, nutritional supplementation and hormone-based fertility management programs for TAI has been developed and implemented successfully. A wider adoption of this technique as a package of practices could be key to improving the reproductive efficiency of buffaloes in Nepal.

Intestinal ganglioneuroma in a neonatal Japanese black calf - Short communication.

A 1-day-old male calf presented with clinical signs of severe progressive abdominal distension. Abdominal radiographic and ultrasonic images revealed several loop-like structures in the small intestine, which were indicative of gas retention. Experimental laparotomy was performed. However, the calf died during surgery. At necropsy, a round, well-circumscribed mass (3 × 3 × 2.5 cm) was found in the jejunal wall, and the jejunal lumen was narrowed. The mass was firm and had white to grey appearance on the cut surface. Histologically, the submucosa and the muscle layer were diffusely thickened due to abundance of neural tissues comprising several fascicles of nerve fibres and large aggregates of ganglion cells. Some ganglion cells contained basophilic Nissl substances in their cytoplasm. Immunohistochemically, these cells were positive for S-100 and synaptophysin. Ultrastructural examination revealed that the neoplastic ganglion cells contained dense core vesicles in the cytoplasm. Based on these findings, the neoplastic lesion was diagnosed as ganglioneuroma in the jejunum.

Yarsagumba is a Promising Therapeutic Option for Treatment of Pulmonary Hypertension due to the Potent Anti-Proliferative and Vasorelaxant Properties.

Background and objectives: Pulmonary hypertension (PH) is characterized by the vasoconstriction and abnormally proliferative vascular cells. The available allopathic treatment options for PH are still not able to cure the disease. Alternative medicine is becoming popular and drawing the attention of the general public and scientific communities. The entomogenous fungus Yarsagumba (Cordyceps sinensis) and its biologically active ingredient cordycepin may represent the therapeutic option for this incurable disease, owing to their anti-inflammatory, vasodilatory and anti-oxidative effects. Methods: In this study, we investigated whether Yarsagumba extract and cordycepin possess anti-proliferative and vasorelaxant properties in the context of PH, using 5-bromo-2'-deoxyuridine assay and isolated mice lungs, respectively. Results: Our results revealed that Yarsagumba extract and its bioactive compound cordycepin significantly attenuated the proliferation of human pulmonary artery smooth muscle cells derived from donor and PH subjects. In isolated murine lungs, only Yarsagumba extract, but not cordycepin, resulted in vasodilatation, indicating the probable existence of other bioactive metabolites present in Yarsagumba that may be responsible for this outcome. Conclusion: Future comprehensive in vivo and in vitro research is crucially needed to discover the profound mechanistic insights with regard to this promising therapeutic potency of Yarsagumba extract and to provide further evidence as to whether it can be used as a strategy for the treatment of PH.

Influence of transfixation pinning and casting (TPC) configurations on treatment outcomes in bovine tibial fracture.

The aim of this study was to examine the treatment outcomes of tibial fracture using different configurations of transfixation pinning and casting (TPC) in eight cattle. After pins were inserted to stabilize the fracture site, cast material was placed from the level of the stifle to the hoof (full TPC) in six cases and from the stifle to the proximal metatarsus (partial TPC) in two cases. Five cattle (three full TPC and two partial TPC cases) underwent TPC removal 36-86 days after surgery and resumed productivity. The other three cattle which underwent full TPC suffered from irreversible orthopedic complications within 5 weeks of surgery, while a prolonged healing process was observed in the partial TPC cases. Although TPC is an effective method for treating bovine tibial fracture, full TPC may result in a guarded prognosis in some cattle. However, partial TPC can delay healing. Surgeons should choose the optimal TPC configuration while considering the various factors that affect the healing process.

Correction to: Antibiotic resistance pattern and virulence genes content in avian pathogenic Escherichia coli (APEC) from broiler chickens in Chitwan, Nepal.

The original article [1] contains errors in author panels and their contributions, errors in both the Methodology and the Results sections, and errors with respect to funding sources. The affected sections of the manuscript and their respective regions of corrected text can be viewed ahead.

Changes in the plasma levels of several bone markers in newborn calves during the first two days of life.

The fluctuations in the plasma levels of several bone markers were investigated in newborn calves. Experiment 1 monitored the postnatal changes in the plasma levels of tartrate-resistant acid phosphatase isoform 5b (TRAP5b), total alkaline phosphatase (t-ALP) and bone-specific alkaline phosphatase (BAP) in four calves. These markers increased significantly from 9-20 hr after the first colostrum-suckling compared with the values immediately after birth. Experiment 2 evaluated changes in the plasma TRAP5b, t-ALP, BAP and type I collagen cross-linked N-telopeptide (NTx) levels within 2 days post-birth in five calves with successful passive immunization via colostrum (non-deficient group) and five others with poor colostrum intake (deficient group). The non-deficient group had significantly higher plasma levels of the four parameters around 12 hr of life compared with the deficient group. The results suggest that the increase in plasma bone markers in calves in the first day of life is related to the colostrum intake.

Persistence of uterine bacterial infection, and its associations with endometritis and ovarian function in postpartum dairy cows.

We investigated the relationship between the persistence of uterine bacterial infections with cytologically determined endometritis and ovarian function in 65 postpartum Holstein cows. Vaginal mucus discharges were collected, and endometrial smear samples (n = 130) were collected for cytological and bacteriological examinations from the cows at weeks 5 and 7 postpartum (pp). Blood samples were collected at weeks 3, 5 and 7 pp to determine plasma progesterone concentrations to monitor ovarian activity. According to the bacteriological examination, cows were classified into four groups. The first group (n = 32; 49%) comprised cows negative for bacteria at weeks 5 and 7 pp. The second group (n = 11; 17%) comprised cows with bacterial infections at week 5 pp but that were clear of infection at week 7 pp. The third group (n = 12; 19%) comprised cows without bacteria at week 5 pp but that acquired an infection by week 7 pp. The fourth group (n = 10; 15%) comprised cows with bacterial infections at weeks 5 and 7 pp (persistence of infection). A positive correlation (P < 0.001) was noted between the severity of cytologically determined endometritis, purulent vaginal discharge and the persistence of infection. Cows with persistent infections had a significantly (P < 0.01) prolonged luteal phase compared with cows without infection. In conclusion, the prevalence of cytologically determined endometritis and prolonged luteal phase were significantly increased in cows with persistent infections.

Outpatient treatment for humeral fractures in five calves.

Humeral fractures were treated in 5 calves using unilateral external fixation with epoxy putty fixator (type I). The surgeries were performed under sedation and analgesia, and it involved application of ultrasound-guided brachial plexus block. The surgical procedures were completed in 60 to 90 min, and each calf was returned to the farm on the same day. The fixation allowed each calf to remain with the dam and suckle without strict stall rest and was removed 11 to 62 days post-surgery. The clinical sign of diminished radial nerve function disappeared 40 days to 4 months post-surgery. These observations suggest that this repair technique represents a feasible outpatient treatment for humeral fractures in calves.

Molecular phylogenetic identification of Fasciola flukes in Nepal.

Eighty-one Fasciola flukes collected from 8 districts in Nepal were analyzed for their species identification on the basis of their spermatogenic status and nuclear ribosomal internal transcribed spacer 1 (ITS1) and for their phylogenetic relation with Fasciola flukes from other Asian countries on the basis of the mitochondrial NADH dehydrogenase subunit 1 (nad1) gene. Sixty-one flukes (75.3%) were aspermic Fasciola sp., and 20 flukes (24.7%) were identified as Fasciola gigantica. All of the aspermic flukes displayed the Fh/Fg type in ITS1, which was predominant in aspermic Fasciola sp. from China, and most (60 flukes) displayed the Fsp-ND1-N1 haplotype in the nad1, which had an identical nucleotide sequence to the major haplotype (Fg-C2) of the aspermic flukes from China. These results suggest that aspermic Fasciola sp. was introduced into Nepal from China. Furthermore, the results of the diversity indices, neutrality indices, and median-joining network analysis with reference haplotypes from Asian countries suggest that aspermic Fasciola sp. rapidly expanded its distribution. In contrasts, F. gigantica displayed 10 nad1 haplotypes, which showed higher population diversity indices than the haplotypes of aspermic flukes, indicating that the F. gigantica population was clearly distributed in Nepal earlier than the aspermic Fasciola population. Although the F. gigantica haplotypes from Nepal formed a star-like phylogeny consisting of a main founder haplotype (Fg-ND1-N1), together with some F. gigantica haplotypes from Myanmar and Thailand, the Nepal population differed genetically from F. gigantica populations of neighboring countries as each country had distinct founder haplotype(s).

Diurnal variation of plasma bone markers in Japanese black calves.

To evaluate diurnal variation of plasma bone markers, blood samples were collected from five calves at 2-hr intervals throughout a 24-hr period. Tartrate-resistant acid phosphatase isoform 5b (TRAP5b), carboxy-terminal collagen crosslinks of type-I collagen (CTX), hydroxyproline, bone specific alkaline phosphatase (BALP) and osteocalcin were measured. Cosinor analysis showed a significant rhythm in all bone markers. The acrophase of each bone marker appeared from the early to late morning. The percentage ratio of the amplitude to mesor and the within-subject variability for CTx and osteocalcin were significantly larger than those for TRAP5b and BALP. This marked diurnal variation in five bone markers suggested that the time of blood sampling should be fixed when studying bone marker concentrations in bovine plasma.

Effects of treatment for anestrus in water buffaloes with PGF2α and GnRH in comparison with vitamin-mineral supplement, and some factors influencing treatment effects.

The effect of treatment for anestrus in buffaloes with a PGF2α or GnRH injection and vitamin-mineral (Vit-M) supplementation for 1 to 2 months and some factors influencing the treatment effect were studied. In anestrus buffaloes with CL, an injection of PGF2α tended to show higher estrus detection and pregnancy rates within 17 days after treatment than Vit-M supplementation (P<0.10). In those with inactive ovaries, effect of GnRH and Vit-M did not differ. Body condition score of the animals before treatment affected pregnancy rate within 17 days after treatment (P<0.05). Pregnancy rate within 4 months after treatment was adversely influenced by low serum concentrations of calcium (P<0.01) and gastrointestinal parasitic infection before treatment (P<0.05).

Fluctuation in plasma bone metabolic markers in multiparous and primiparous Holstein cows during early to peak lactation.

Because the role of bone turnover during early to peak lactation in dairy cows is unclear, plasma levels of osteoclast- or collagen-specific bone resorption and osteoblast-specific bone formation markers from postpartum week 3 (W+3) to W+7 were compared with prepartum W-3 levels in multiparous (n = 15) and primiparous (n = 13) Holstein cows. The multiparous group showed a consistently low level of osteoclast-specific bone resorption markers, while collagen-specific resorption and osteoblast-specific formation markers decreased at postpartum W+7 and from W+5, respectively. In the primiparous group, the plasma levels of all three markers decreased from postpartum W+3. These results suggest that the calcium demand during the transition from early to peak lactation in dairy cows is less affected by bone turnover.

A distinct genetic population of Gongylonema pulchrum from water buffaloes in Nepal.

Whole-length esophagi of 111 Murrah cross water buffaloes (Bubalus bubalis) were collected in the Kathmandu and Chitwan districts of Nepal from December 2009 to February 2010. Gullet worms showing a typical epithelium-dwelling character were detected in 13 of 53 (24.5%) buffaloes in Kathmandu and in 5 of 58 (8.6%) buffaloes in Chitwan. The worms' morphology and measurements were identical to those of Gongylonema pulchrum Molin, 1857, except for the length of the left spicules relative to the body length. Scanning electron microscopy did not detect any further morphological differences regarding the collected specimen from Nepal compared with G. pulchrum . The ribosomal RNA gene (rDNA), including internal transcribed spacer (ITS) 1 and 2, and a partial region of the cytochrome c oxidase subunit I (COI) of mitochondrial DNA of the worms were characterized and compared with those of G. pulchrum collected from cattle, deer, wild boars, and monkeys in Japan and from cattle in Iran. The 18S, 5.8S, and 28S rDNA nucleotide sequences of the buffalo-collected worms had 99.8% (1,779/1,782), 100% (158/158), and 98.3-98.8% (3,494-3,507/3,551) identities, respectively, with those of G. pulchrum from the other host mammals. The ITS regions exhibited higher variations between the buffalo-collected worms and G. pulchrum from the other host mammals (85-88% identity for ITS1 and 56-80% identity for ITS2). The COI also showed lower identities (89.2-90.2%), although only a single amino acid substitution was noted compared with the majority of G. pulchrum samples collected in Japan. Based on these molecular genetic characters in the rDNA and COI mitochondrial DNA, together with a shorter left spicule length relative to body length, the gullet worms isolated from buffaloes in Nepal might belong to a distinct local or buffalo-preferring population of G. pulchrum, although its geographical distribution on the continent and host specificity remain to be clarified.

Mycoplasma infection in the uterus of early postpartum dairy cows and its relation to dystocia and endometritis.

This study investigated the incidence of mycoplasma infection in the uterus of postpartum Holstein dairy cows and its relationship to the occurrence of endometritis. The genital tracts of 209 cows from three dairy farms in the Iwate Prefecture, Japan, were examined at Weeks 5 and 7 postpartum. The condition of the cervicovaginal mucus was assessed using a Metricheck device and assigned a score from 0 (clear mucus) to 4 (purulent material with fetid odor). Intrauterine samples (N = 418) were collected at Weeks 5 and 7 postpartum using a cytobrush. After its withdrawal, swab samples were placed in mycoplasma culture broth at 37 °C for 72 hours. A novel and rapid polymerase chain reaction was used to detect seven mycoplasma species (Mycoplasma bovis, M. arginini, M. bovigenitalium, M. californicum, M. bovirhinis, M. alkalescens, and M. canadense). The cytobrush was also rolled gently along the length of a glass slide for subsequent polymorphonuclear neutrophil count. The diagnostic criteria for cytological endometritis were 6% or more and 4% or more polymorphonuclear neutrophils at Weeks 5 and 7, respectively. From a subset of cows, additional swabs were rolled against the cytobrush and then placed in transport medium. These samples were then plated on specific agar plates and cultured under aerobic and anaerobic conditions to identify other bacteria present. The incidence of dystocia at the last calving was compared in mycoplasma positive and negative cows. Of the seven mycoplasma species, only M. bovigenitalium was detected; it was detected in 31 of the 418 uterine swabs (7.4%). Twenty-four cows were positive for M. bovigenitalium (eight cows at Week 5, nine cows at Week 7, and seven cows at both Weeks 5 and 7). The incidence of dystocia was higher (P < 0.0001) in mycoplasma positive (7/24; 29.2%) compared with mycoplasma negative (4/185; 2.2%) cows. However, there was no significant association between dystocia at last calving and subsequent uterine infection with other bacteria. In addition, the incidence of cytologic endometritis was higher (P < 0.05) in mycoplasma positive (8/16; 50%) than in mycoplasma negative (47/193; 24.4%) cows at Week 7. Therefore, we concluded that M. bovigenitalium infection in the uterus might be associated with recent dystocia and with cytologic endometritis in postpartum dairy cows.

Response of Holstein cows with milk fever to first treatment using two calcium regimens: a retrospective clinical study.

The responses of 64 Holstein cows with milk fever to first treatment with 500 ml of either of 2 intravenous calcium (Ca) solutions, one containing Ca alone (group A, n = 32) or 1 containing Ca, phosphate and magnesium (group B, n = 32), were evaluated by selected clinical signs and serum biochemical data. Based on the cow's ability to stand, treatment response was categorized into "immediate response" (stood after single treatment), "delayed response" (stood after repeated treatments) and "non-response" (slaughtered despite repeated treatments). No significant differences among the response categories were found between the two groups, suggesting that the solution containing Ca borogluconate alone was sufficient for the first treatment of milk fever.

A novel technique of ultrasound-guided brachial plexus block in calves.

An interventional ultrasound technique to increase the safety of surgical treatment of the calf forelimb was tested. First, the brachial plexus was evaluated using ultrasonography and then 2% lidocaine was injected under ultrasound guidance. Ultrasonically, the brachial plexus appeared as multiple hypoechoic areas surrounded by a hyperechoic rim or a hyperechoic structure characterised by multiple discontinuous lines. It was located between the omotransverse muscle and axillary artery and vein. The sensitive effect in the forelimb was seen mainly in the area supplied by the musculocutaneous nerve, indicating successful blockage in the nerve plexus. Out of the eight forelimbs, the motor effect was observed in seven forelimbs. These results suggest the clinical feasibility of ultrasound-guided brachial plexus block in bovine medicine, although further studies are needed to examine various approaches, including the sites of needle insertion and the appropriate volume and dosage of anaesthetic.

Relationship between sex hormone fluctuations and biomarkers of bone resorption in bovine plasma during the oestrous cycle.

The objective of this study was to determine the potential influence of fluctuations in the sex hormones progesterone and oestradiol-17ß (E(2)), on biomarkers of bone resorption (hydroxyproline [HYP] and tartrate-resistant acid phosphatase isoform 5b [TRAP5b]) during the oestrous cycle of Holstein cows. Over the course of the study, plasma HYP concentrations did not change and alterations in the concentration of TRAP5b negatively correlated with E(2) levels: enhanced TRAP5b activity correlated with decreased E(2) concentrations below a defined level. This finding enhances the understanding of calcium homeostasis in post-partum dairy cows.

Colorimetric assay for the quantification of serum iodixanol concentration and its application on estimation of glomerular filtration rate in cattle.

A colorimetric assay used to quantify the non-ionic contrast medium iodixanol in sera was validated and compared with high-performance liquid chromatography. The application of this assay to estimate glomerular filtration rates (GFR) in cattle was examined. Serum iodixanol was de-iodinated by alkaline hydrolysis and the amount of released iodine was subsequently determined using a ceric arsenite method. There was a close correlation between the two methods using identical specimens. In clinically healthy cattle with different body weights, the reference value (166.3-178.8 mL/min/m(2)) based on body surface area was fairly stable as compared with that (2.13-3.63 mL/min/kg) based on body weight. Based on GFR data in healthy and renal-impaired cattle, when the GFR decreased to more than 60% of the reference value, serum urea nitrogen and creatinine concentrations increased. The colorimetric assay is a simple method for the estimation of GFR in cattle and requires no expensive equipment.

Dose-response effects of an aqueous formulation of calcitriol and evaluation of renal function in nonpregnant cows.

Dose responses of plasma calcitriol, calcium (Ca), bone metabolic markers and glomerular filtration rate (GFR) were evaluated in four nonpregnant Holstein cows treated subcutaneously with an aqueous formulation of calcitriol at four doses in a 4 × 4 Latin-square design. Calcitriol, Ca, and markers of bone metabolism were analyzed in plasma samples. GFR was measured in predose and day 5 samples. Plasma calcitriol and Ca concentrations increased dose-dependently. The calcitriol dose was positively correlated with the area under the concentration-time curve of plasma calcitriol. Bone formation markers tended to increase from day 3 onward for all doses. No significant changes in GFR were noted. Thus, exogenous calcitriol administered between 0.0625 and 0.5 µg/kg body weight elicited dose-dependent increases in both plasma calcitriol and Ca and elevated bone formation markers without affecting renal function in nonpregnant cows.