Isolation and characterization of an Indian ORF virus from goats

Isolation and characterization of an orf virus has been described here. The virus was isolated from an outbreak of 'scabby mouth' in goats in Northern India. Viral morphology from the scab biopsy revealed typical ovoid-shaped particles characteristic of Parapoxvirus. Virus was isolated from sonicated scab suspension and characterized by restriction enzyme (RE) analysis and sequencing of full-length GM-CSF- and interleukin-2 inhibitory factor (GIF) gene. RE pattern of the virus did not show close resemblance to most of the orf viruses published earlier. However, it showed high sequence identity and closer phylogenetic relationship with previously published ORFV-SA00 strain, as evident from the nucleotide and deduced amino acid sequence of GIF gene.     

Effect of cattle-specific estrus molecules on libido and semen production of zebu bulls under tropical climate

Zebu bulls are a shy breeder and they exhibit optimum libido in the presence of females with estrus phase. Continuous semen collection with the use of male dummy leads to lack of adequate sexual stimulation. Therefore, the present study was designed to test the effect of estrus-specific molecule(s) for effective sexual preparation of donor bulls. The bulls were divided into normal and poor libido group, five bulls in each group by taking 1-month control study data after collecting the information of individual bull’s sexual behaviour during semen collection by regular semen collector. The bulls were never being exposed to female animals and semen was collected by an artificial vagina. The ten animals were exposed to a glycerol-water solution (50/50 v:v) as control and then exposed to estrus-specific molecules one by one. The estrus-specific molecules like squalene, 1-iodoundecane, acetic acid, coumarin, propionic acid, oleic acid, and 2-butanone were purchased from Sigma-Aldrich Company, USA, and the molecules were solubilised individually in a non-pressurised aerosol dispenser as 1.0% concentration in glycerol-water solution (50/50, v:v). Identical bulls were used as the control and exposed to each molecule one by one by giving a refractory period of 14 days. A nasal spray of acetic acid or 2-butanone significantly (p < 0.05) reduced reaction time (RT) and total time taken to ejaculate (TTTE) in normal libido bull group. Semen volume, sperm concentration, and the total number of sperm per ejaculation obtained did not show significant improvement in the normal libido group of bulls after the application of estrus-specific molecules as compared to the control. In poor libido group, acetic acid, oleic acid, and 2-butanone application showed significant (p < 0.01) improvement in RT and TTTE as compared to the control group, whereas semen production variables like sperm concentration and total sperm output per ejaculation increased significantly (p < 0.05) except semen volume. There was significant (p < 0.01) reduction in RT (%) and TTTE (%) after the application of acetic acid followed by 2-butanone and oleic acid. The sperm concentration and total sperm output per ejaculation were more after the application of each molecule but significant increase (p < 0.05) in sperm concentration was observed with 2-butanone (11.42%), acetic acid (11.42%), and oleic acid (10.13%), whereas total sperm output per ejaculation increased significantly (p < 0.05) only after the application of acetic acid and 2-butanone (24.75% and 26.84%). Hence, it can be concluded that acetic acid, 2-butanone, and oleic acid are effective for better sexual preparation of Sahiwal bulls and total sperm output per ejaculation.

     

Molecular cloning and expression of FGF2 gene in pre‐implantation developmental stages of in vitro‐produced sheep embryos

Early embryonic mortality is one of the main sources of reproductive loss in domestic ruminants including sheep. Fibroblast growth factor‐2 (FGF‐2) is a member of FGFs family that mediates trophoblast activities and regulates embryonic development in various species. In this study, we have cloned, characterized sheep FGF2 cDNA (KU316368) and studied the expression in sheep embryos. Ovaries of non‐pregnant sheep were collected from local abattoir and matured in culture medium at 38.5ºC, 5% CO₂, 95% humidity for 22–24 hr. The matured oocytes were inseminated with capacitated spermatozoa in Brackett and Oliphant medium and resulted embryos were cultured in CO₂ incubator for 6–7 days to complete the developmental stages from two cells to blastocyst stage. Total RNA was extracted from immature oocytes (n = 100), mature oocytes (n = 100) and different stages of embryos such as 2 cell (n = 50), 4 cell (n = 25), 8 cell (n = 12), 16 cell (n = 6), morula (n = 5) and blastocyst (n = 3). The total RNA isolated from the oocytes and embryos was reverse transcribed and subjected to real‐time polymerase chain reaction using sequence‐specific primers and SYBR green as the DNA dye. On sequence analysis, the nucleotide sequence of sheep FGF2 exhibited highest sequence similarity with cattle (100%) and least with rat and mouse (69.2%). At the deduced amino acid level, a highest degree of similarity was noticed with cattle, buffalo, goat, pig, camel and horse (100%) and lowest degree of identity with rat, human and mouse (98.2%). The FGF2 mRNA expression was higher in immature and mature oocytes and gradually decreases from 2‐cell stage of embryo to the blastocyst stage. More over a significant differences in FGF2 mRNA expression (p < .05) were observed between immature oocytes and all pre‐implantation stages of embryo. It can be concluded that FGF‐2 plays a significant role in pre‐implantation and early development of embryos in sheep.     

Effects of long-term growth hormone-releasing factor treatment on growth, feed conversion efficiency and dry matter intake in growing female buffaloes (Bubalus bubalis)

Effects of long-term growth hormone-releasing factor (GRF) on growth performance, feed conversion efficiency (FCE) and dry matter intake (DMI) were studied in growing buffaloes. Twelve female Murrah buffaloes of 6-8 months of age were divided into two groups of six each on the basis of their body weights so that the average body weights of the groups did not differ (p > 0.05). Animals of each group were administered intravenously with either sterile distilled water (control group) or equal volume of GRF solution containing 10 mug GRF (1-44)-NH2/100 kg body weight (treatment group) at fortnight interval from week 6 (5-week pre-treatment period) for 36 weeks (weeks 6-42 treatment period). Thereafter a 10-week post-treatment period was added. All the animals were weighed consecutively 2 days in a week and the average body weight of the two observations in the week was thus considered for further calculation. Dry matter intake was recorded daily. Average daily gain, FCE and DMI/100 kg body weight were also calculated. Plasma progesterone was estimated in the samples collected twice a week at 3-4-day intervals to assess whether either group had begun ovarian cycles. It was found that ADG and FCE were higher (p < 0.01) in GRF-treated animals during treatment and even 10-week post-treatment period. Interestingly, total DMI was not different (p > 0.05) between the groups during treatment period but found to be lower in GRF treated animals during post-treatment period. The DMI/100 kg body weight was lower (p < 0.01) in GRF-treated animals during treatment and even after cessation of treatment for 10 weeks. The GRF administration for long-term increased (p < 0.05) plasma progesterone. Plasma progesterone concentrations suggest that no animal from either group reached puberty till the end of the experiment. In conclusion, repeated GRF administration for longer term decreased (p < 0.01) DMI/100 kg body weight and increased (p < 0.01) FCE and enabled the animals to grow faster.     

Effects of Long-term Growth Hormone-releasing Factor Administration on Plasma Growth Hormone, Luteinizing Hormone and Progesterone Profiles in Growing Female Buffaloes (Bubalus bubalis)

To investigate the effects of long-term growth hormone-releasing factor (GRF) administration on plasma growth hormone (GH), LH and progesterone and body weight gain in growing buffalo calves, 12 female Murrah buffaloes within the age group of 6-8 months of age were divided into two groups (treatment and control groups) of six each in such a way so that average body weights between the groups did not differ (p > 0.05). Control buffaloes were not given any hormonal treatment and treatment group buffaloes were treated with synthetic bovine GRF [bGRF (1-44)-NH(2)] at the rate of 10 microg/100 kg body weight intravenously at an interval of 15 days from week 6 (5-week pre-treatment period) till 18 injections were completed (week 6-42 treatment period) and thereafter, effect of exogenous GRF were observed for 10-week post-treatment period. Jugular blood samples were drawn twice a week at 3-4-day intervals for plasma GH, LH and progesterone quantification. Body weight of all animals was recorded twice a week. During pre-treatment period, mean plasma GH, LH and progesterone did not differ (p > 0.05) between the groups. But during treatment as well as post-treatment period, mean plasma GH levels were found to be significantly (p < 0.01) higher in treatment than control group of buffaloes. Administration of GRF for longer term sustained a higher level of plasma GH even after cessation of treatment. GRF-treated buffaloes attained higher (p < 0.01) body weight than the controls. Repeated GRF administration for long-term significantly (p < 0.01) increased plasma LH and progesterone. In conclusion, repeated long-term exogenous GRF administration induces and even enhances GH release without any sign of refractoriness. GRF may, therefore, be used to induce daily GH release without loss of responsiveness over an extended period of time in young growing female buffaloes and it may assist these animals to grow faster.     

Effect of feeding Lagerstroemia speciosa and conventional fodder based rations on nutrient utilization,ruminal metabolites and body weight gain in mithun (Bos frontalis)

The aim of the present study was to evaluate the effect of feeding green fodder, rice straw and concentrate‐based total mixed rations (TMR) on dry matter (DM) intake (DMI), nutrient utilization, rumen fermentation patterns and body weight (BW) gain (BWG) in mithun (Bos frontalis) calves. In a randomized block design, male mithun calves (n = 18, 8–10 months of age, 121 ± 2 kg BW) were randomly divided into three experimental equal groups (six animals in each group) and fed isonitrogenous TMRs ad libitum for 120 days. The TMR₁ contained 30% Napier grass and 30% rice straw, TMR₂ contained 60% rice straw and TMR₃ contained 30% tree leaves (Lagerstroemia speciosa) and 30% rice straw (DM basis). All the TMRs contained 40% concentrate mixture (DM basis). The results indicated that the BWG, DMI and feed conversion efficiency were significantly (p < 0.01) increased with the inclusion of green fodder in TMRs. The apparent digestibility of DM, crude protein, ether extract, crude fibre and nitrogen free extract were also improved significantly (p < 0.01) with the inclusion of green fodder in TMRs. The higher concentration of total nitrogen and total volatile fatty acid in rumen liquor, but low ruminal pH were evident in animals fed green fodder supplemented TMRs. An increased (p < 0.01) molar proportion of acetic acid was evident in animals fed rice straw‐based TMR. In contrast, the molar proportion of propionic and butyric acids were significantly (p < 0.01) higher in animals fed green fodder supplemented TMRs. On the basis of higher DMI and higher daily BWG, it is concluded that Napier grass and L. speciosa tree leaves may be incorporated upto 30% (DM basis) in TMR of growing mithuns for feeding in complete confinement system.     

Polyphasic phenotypic and genetic analysis reveals clonal nature of Xanthomonas axonopodis pv. punicae causing pomegranate bacterial blight

Yellow-pigmented bacteria isolated from blight-affected pomegranate leaves and fruit across seven Indian states in epidemics during the years 2008–2016 were characterized and identified using phenotypic and genotypic tools. All bacterial isolates shared phenotypic traits such as colony morphology, NaCl and pH sensitivity and fuscan production, and caused typical lesions on pomegranate plants upon artificial inoculation. Analysis of 16S ribosomal DNA and 16S–23S rDNA intergenic spacer sequences confirmed their identity as Xanthomonas axonopodis pv. punicae. The new isolates collected after 2000 were compared with an old isolate from the 1950s using polyphasic taxonomic approaches including multilocus sequence analysis (MLSA). Nucleotide polymorphism in 24 isolates for nine genomic loci (dnaK, fyuA, gyrB (Young), gyrB (Almeida), rpoD, fusA, gapA, gltA and lepA) showed minor variations in loci fyuA and gyrB. Isolates were grouped into four nearly identical sequence types, ST1, ST2, ST3 and ST4, based on their allelic profiles, ST3 being widespread in Indian states. Molecular phylogenetic analysis of concatenated 5690 bp with other Xanthomonas pathovars revealed its close genetic similarity with the X. citri group. The blight outbreak in diverse geographical locations is attributed to a re-emerged clonal population of X. axonopodis pv. punicae on a genetically homogenous pomegranate cultivar. The latently infected vegetative planting material of elite pomegranate cultivars contributed to the dissemination of the bacterial inoculum. This study highlights and forewarns of the role played by the clonally propagated elite pomegranate cultivars in disseminating and sustaining clonal populations of this bacterial plant pathogen in many Indian states.     

Oestrous Behaviour and Timing of Ovulation in Relation to Onset of Oestrus and LH Peak in Mithun (Bos frontalis) Cows

The objectives of the study were to evaluate the oestrus behaviour and to determine the timing of ovulation in relation to onset of oestrus and the pre-ovulatory LH surge in mithun (Bos frontalis). For this purpose, the blood samples collected at 15-min intervals for 9 h period following onset of oestrus and thereafter, at an interval of 2 h till 4 h post-ovulation for three consecutive cycles from 12 mithun cows were assayed for plasma LH and progesterone. Ovulation was confirmed by palpation of ovaries per rectum at hourly intervals. Various signs of behavioural oestrus were also recorded. The common signs of oestrus and their frequency of occurrence in mithuns were following and mounting by male mithuns (100%), standing to be mounted (100%), frequent urination (62.33%), raising of tail (65.23%), swelling of vulva (54.26%) and congestion of vulvar mucous membrane (69.87%). The pre-ovulatory LH surges consisted of several pulses (2.92 +/- 0.26 pulses/animal; range, 1-4). The mean (+/-SEM) peak level of LH for individual mithun varied from 6.99 +/- 0.44 to 12.69 +/- 2.10 ng/ml and the mean pooled LH peak concentration was 9.10 +/- 0.60 ng/ml. The highest peak (highest amplitude of LH during LH surge) was 10.83 +/- 0.76 ng/ml (range, 8.07-16.49 ng/ml). The duration of LH surge was 6.98 +/- 0.22 h (6-8 h). Onset of LH surge was at 1.23 +/- 0.17 h post-oestrus onset (range, 0.25-2.25 h). Mean plasma progesterone stayed low (<0.24 ng/ml) during the entire duration of sampling. Ovulation occurred at 26.92 +/- 0.31 (range, 26-29 h) after the onset of oestrus and 18.63 +/- 0.35 h (range, 17-20.75 h) after the end of LH surge. The occurrence of the highest LH peaks within a narrow time frame of 2- to 5-h post-oestrus onset in mithuns could have contributed to the animals ovulating within a narrow time interval. These results are very promising from a practical standpoint of potential success when AI program in this species is implemented in a big way. Furthermore, the results of the occurrence of LH pulses during pre-ovulatory LH surges, which are required for ovulation in this species of animals, is unique and species specific.     

Isolation and characterization of a novel antigenic subtype of infectious bronchitis virus serotype DE072.

Three infectious bronchitis virus (IBV) isolates, CU82792, CU82805, and CU82808, were recovered from sentinel chickens placed with three different layer flocks of a large commercial poultry farm in New York State. The three isolates were classified as members of the DE072 serotype on the basis of 1) their S1 genes could be amplified with only a modified primer designed for the DE072 serotype and 2) their restriction fragment length polymorphism patterns, after digestion with endonucleases HaeIII, BstyI and XcmI, were indistinguishable from that of DE072 virus. Additional characterization of one of the isolates, CU82805, revealed that its S1 gene bears approximately 96% identity at the nucleotide level and 94% identity at the amino acid level with DE072. Yet, in an in vitro reciprocal virus neutralization test, only a one-way neutralization was observed, i.e., antiserum to CU82805 neutralized DE072, whereas CU82805 was not neutralized by DE072 antiserum. Implications of these findings with regard to IBV diagnosis and immunization are discussed.