Comparative antimicrobial activity in Murrah buffalo (Bubalus bubalis) Heifers’ urine and urine distillate

Background:Murrah buffalo is a breed of water buffalo(Bubalus bubalis)reared for milk and meat,especially in Northern India.There are so many studies on the antimicrobial potential of the cow(Bos indicus)urine but buffalo urine has rarely been studied.This study was aimed to evaluate the antimicrobial activity in whole buffalo urine and its distillate.Methods:Urine specimens were collected from seven Murrah buffalo heifers in the morning over three days from all seven heifers and distillated to prepare urine distillate(UD).The antimicrobial activity was determined through 96 well microplate method diluting(1:1)urine and UD in Mueller Hinton liquid medium against 919 microbial strains belonging to 148 species of 49 genera(Candia,11 genera of Gram+ve bacteria,37 genera of Gram-ve bacteria).The test strains were taken from the repository,revived and tested for purity before testing.The test strains were of clinical origin(372),environmental origin(496)and also from reference strain repositories(51).Results:Of the 919 test strains,a total of 57.89%and 56.04%of the tested strains were susceptible to UD and urine,respectively.There was a no significant(p>0.4)difference in the antimicrobial activity of UD and urine against 919 strains of microbes,regardless of their genus,species,and Gram staining reaction.Similarly,there was no significant(p>0.4)difference between the susceptibility of bacterial and Candida strains to UD or urine.However,strains of clinical origin were more often resistant to buffalo UD and urine(p<0.01)than strains isolated from environmental sources irrespective of their Gram staining characteristics(p>0.05).A significantly(p<0.01)higher proportion of reference strains was susceptible to UD and urine than microbial strains of clinical and environmental origin.For buffalo urine and UD,the most susceptible strains were Stenotrophomonas maltophilia(13/13)followed by Citrobacter freundii(8/10);Salmonella enterica ssp.enterica(21/27),Geobacillus stearothermophilus(23/30),Staphylococcus aureus(13/17),and Paenibacillus larvae(7/10)strains.The most resistant strains belonged to Acinetobacter calcoaceticus(15/20)species followed by strains of Paenibacillus alvei(11/16),Edwardsiella tarda(20/30),Bacillus megaterium(7/11)and Escherichia coli(59/110)species.There was insignificant(p,>0.05)difference in susceptibility of strains of different species of the same genus;however,among all the Bacillus species strains of B.coagulans were the most susceptible(66.7%)and strains of B.megaterium were the least susceptible(36.4%),similarly Enterococcus faecium strains were more susceptible(68.2%)than strains of Enterococcus faecalis(38.9%),70%strains of P.larvae were susceptible to urine and UD but only 31.3%of strains of P.alvei were susceptible.Strains of Klebsiella pneumoniae and Raoultella terrigena,earlier belonging to the same genus Klebsiella,had comparable susceptibility to buffalo urine and UD.Conclusion:The study concluded that buffalo urine may be a potential antimicrobial and may be explored further for identification of active antimicrobial compounds in buffalo urine.

Detection of Babesia bovis and Babesia bigemina in Water Buffaloes (Bubalus bubalis) in Endemic Areas of São Paulo State, Brazil

Babesiosis is a tick-transmitted disease that causes severe economic losses to the cattle industry in Brazil. Water buffaloes (Bubalus bubalis) are often carriers of Babesia spp., but there are no studies that provide an accurate estimation of this infection in animals raised in regions of endemic stability. This study was conducted to investigate Babesia bovis and B. bigemina infections in 108 water buffaloes (50 calves and 58 adult females) located in areas of S&atildeo Paulo state, where the animals were continuously exposed to Rhipicephalus microplus ticks. B. bovis and B. bigemina infections were screened by microscopic examination of blood smears, nested PCR (nPCR) and quantitative real-time PCR (qPCR), which were also used to estimate the number of copies (NC) of the cytochrome b (mt-cytB) gene in the blood samples. B. bigemina was found in blood smears of three calves from Alambari herd (all with less than 0.1% parasitemia). Molecular techniques were more sensitive than blood smears to diagnose piroplasms in water buffaloes: 20.37% and 100.00% for B. bovis-infected animals and 59.26% and 100.00% for B. bigemina-infected animals, respectively for nPCR and qPCR. The NC of mt-cytB gene of B. bovis and B. bigemina in blood samples revealed significant effects (p p ≤ 0.05) for B. bigemina (2.80 ± 0.06) than for B. bovis (2.61 ± 0.05). Within each herd-age, differences between the species’ NC values were found only in Alambari calves, which showed significantly higher (p ≤ 0.05) NC of B. bigemina (3.48 ± 0.13). The calves and cows from Ibaté showed the lowest NC of B. bigemina (2.29 ± 0.13 and 2.63 ± 0.14) and B. bovis (2.54 ± 0.11 and 2.37 ± 0.12), respectively. These data suggest a high prevalence of B. bovis and B. bigemina infection in the buffalo population in endemic areas of S&atildeo Paulo state.

Tissue-specific Temporal Exome Capture Revealed Muscle-specific Genes and SNPs in Indian Buffalo(Bubalus bubalis)

Whole genome sequencing of buffalo is yet to be completed, and in the near future it may not be possible to identify an exome （coding region of genome） through bioinformatics for designing probes to capture it. In the present study, we employed in solution hybridization to sequence tissue specific temporal exomes （TST exome） in buffalo. We utilized cDNA prepared from buffalo muscle tissue as a probe to capture TST exomes from the buffalo genome. This resulted in a prominent reduction of repeat sequences （up to 40%） and an enrichment of coding sequences （up to 60%）. Enriched targets were sequenced on a 454 pyro-sequencing platform, generating 101,244 reads contain- ing 24,127,779 high quality bases. The data revealed 40,100 variations, of which 403 were indels and 39,218 SNPs containing 195 nonsyn- onymous candidate SNPs in protein-coding regions. The study has indicated that 80% of the total genes identified from capture data were expressed in muscle tissue. The present study is the first of its kind to sequence TST exomes captured by use of cDNA molecules for SNPs found in the coding region without any prior sequence information of targeted molecules.

Functional Genes in Relation to Residual Feed Intake in Murrah Buffalo Heifers

High Feed efficiency (FE) in growing heifers has economic importance in dairy, but remains less understood in buffaloes. Feed conversion efficiency is defined as dry matter intake (DMI) per unit body weight gain and is determined as residual feed intake (RFI), i.e., the difference between actual and predicted feed intake to gain unit body weight during a feed trial run for 78 days under control feeding. A large variation was identified ranging between -0.42 to 0.35 in growing buffalo heifers (n = 40) of age between 11 to 15 months. An average daily weight gain (ADG) varied between 382.0 and 807.6 g/day when compared with the control-fed heifers at an organized buffalo farm. The whole blood transcriptome data obtained from the selected growing heifers from extremes of estimated high and low RFI efficiency were compared with the reference assembly generated from the transcriptome of multiparous buffaloes (n = 16) of diverse age of maturity, period of regaining post partum cyclicity and level of milk production. Differentially expressed genes (DEGs) were identified using the reference genome of Mediterranean water buffalo. GO: terms (Padj 0.05, FDR 0.05) enriched by annotated DEGs and biological pathways in gene network for RFI efficiency trait were identified. GO: terms specific to pre-transcriptional regulation of nucleus and Chromatin organization under Nucleoplasm, Energy balancing, Immunity, Cell signaling, ROS optimization, ATP generation through the Electron Transport chain and cell proliferation were determined. The study reveals the indicators targeting the actual metabolic changes and molecular functions underlying the feed utilization capacity of buffaloes. Estimated RFI efficiency revealed a large variation over heifers which may lower the DMI even up to 13.6% thus, enabling an increase in ADG up to 16% by involving efficient heifers in breeding plan. The study revealed a scope of high gain by selective breeding for FE in heifers. FE variants catalogued in the study are useful breed-specific RFI markers for future reference. The study contributes to the understanding of feed efficiency in buffaloes and its association with key interactive traits such as reproduction and growth. This knowledge can be utilized to develop more effective breeding programs.

Influence of Concentrate in the Diet on the Metabolic, Biochemical, and Endocrine Profile of Buffalo Heifers

The objective of this study was to evaluate the profile of the main serum biochemical variables in buffalo heifers receiving diets with different concentrations of concentrate. Twenty buffalo heifers of the Murrah breed, aged six to eight months, were randomly assigned to four groups, with five replicates each, to be adapted to levels of 20%, 40%, 60%, and 80% of concentrate in the diet, composed of sugarcane (bulk) and concentrated feed based on wheat, corn, and soybean meal. The experimental diets were constituted in the proportion “bulk: concentrate”, based on dry matter, of “80:20” (Treatment 1), “60:40” (Treatment 2), “40:60” (Treatment 3), and “20:80” (Treatment 4). Biomarkers were evaluated for energy, protein, mineral, and endocrine profiles, as well as enzymatic activity. A positive linear effect was observed for Final Body Weight. In the energy profile, there was a positive linear effect for total cholesterol and HDL cholesterol and a quadratic effect for plasma glucose. In the protein profile, a quadratic effect was observed for the urea: creatinine ratio and a positive linear effect for total protein and albumin. For the enzymatic activity, only the variable gamma-glutamyltransferase presented a quadratic effect. In the mineral profile, a quadratic effect was observed for calcium levels and a positive linear effect for phosphorus levels and negative for chlorine levels. In the endocrine profile, no statistical differences were observed. Diet with until 80% of concentrate does not negatively influence biochemical parameters that represent the profile of energy, protein, mineral and thyroid hormones for growing buffalo heifers and favors the performance of the animals.

水牛泌乳期与干乳期差异血清生化指标及代谢物研究

本研究旨在比较泌乳期与干乳期水牛(Bubalus bubalis)差异血清生化指标及差异血清代谢物的组成,探讨血清生化指标与代谢物的相互关联,进一步了解水牛营养代谢过程的调控机制。选取泌乳期(泌乳第100~200天)健康水牛9头和干乳期(停乳后第30~60天)健康水牛9头,每头牛采血清样本两份,一份用生化分析仪进行20项指标检测;另一份用超高效液相色谱-串联质谱法(ultra-high performance liquid chromatography-tandem mass spectrometry,UPLC-MS/MS)检测分析水牛血清中内源性代谢物。在生化指标检测结果中,泌乳期的谷草转氨酶/谷丙转氨酶(P<0.05)、甘油三酯(P<0.01)显著低于干乳期的,说明水牛在泌乳期时对酯类物质的利用效率更高;而泌乳期的胆固醇(P<0.01)、低密度脂蛋白(P<0.01)、高密度脂蛋白(P<0.001)、尿素氮(P<0.01)、葡萄糖(P<0.01)和免疫球蛋白A(P<0.0001)等指标显著高于干乳期的,说明泌乳期时水牛的物质代谢更高。对于血清代谢物进行分析得出共有183种代谢产物在2个时期中存在显著差异(P<0.05,VIP>1),这些代谢产物共富集到8条显著不同的代谢途径,分别是D-谷氨酰胺和D-谷氨酸代谢,精氨酸和脯氨酸代谢,色氨酸代谢,卟啉和叶绿素代谢,甘氨酸、丝氨酸和苏氨酸代谢,组氨酸代谢,不饱和脂肪酸的生物合成,以及氮代谢(P<0.05)。这些结果说明在泌乳期时,水牛吸收的营养物质主要用于参与多种氨基酸的合成及脂肪酸的合成与利用。

水牛卵泡不同发育阶段差异表达基因分析

本研究旨在利用转录组测序技术探究摩拉水牛(Bubalus bubalis)发情期不同大小卵泡的基因表达谱,并进一步了解其对卵泡发育过程中相关激素的影响。通过对摩拉水牛进行Ovsynch法同期发情处理,B超检测卵泡大小并按照大、中、小3种类型(>8 mm、4~7 mm、0~3 mm)区分,活体采集卵泡液后提取RNA送单细胞转录组测序。将获得的数据进行组间两两比较,以|log2fold change|≥1且P<0.05为标准筛选差异转录本,并取各组差异表达转录本并集共1473个转录本做表达趋势筛选,其中587个转录本符合牛卵泡发育过程中的转录调控规律,通过GO和KEGG富集分析并筛选得到了33条通路,各通路主要与细胞间相互作用、类固醇生成和激素产生、细胞分化和发育、细胞运动、代谢调节、生理适应性等过程相关。通过对通路上的基因进行蛋白质互作网络分析,进一步获得了可能影响水牛卵泡发育过程中激素调节的关键基因,如BAMBI、FST、INHA、INHBA、INHBB、GATA6和GFAP。综上,该研究结果为进一步探究水牛卵泡发育过程中的关键基因及可能的分子调控机制提供了参考。