Impact of probiotic administration on the health and fecal microbiota of young calves: a meta-analysis of randomized controlled trials of lactic acid bacteria

Before weaning, dairy calves are susceptible to many pathogens which can affect their subsequent performance. The use of lactic acid bacteria (LAB) has been identified as a tool to maintain the intestinal microbial balance and to prevent the establishment of opportunistic pathogenic bacterial populations. However, a consensus has not been reached as to whether probiotics may be effective in reducing the prevalence of gastrointestinal diseases in young calves. The aim of this meta-analysis was to assess the effect of probiotics on diarrhea incidence and the intestinal microbial balance. LAB supplementation has been shown to exert a protective effect and to reduce the incidence of diarrhea (relative risk, RR=0.437, 95% confidence interval (CI) 0.251-0.761). In the subanalysis, this protective effect of the probiotics against diarrhea was observed only in trials that used whole milk (RR=0.154, 95% CI 0.079-0.301) and trials that used multistrain inocula (RR=0.415, 95% CI 0.227-0.759). Probiotics did not improve the fecal characteristics (standardized mean difference, SMD=-0.4904, 95% CI -1.011-0.035) and were unable to change the LAB:coliforms ratio (SMD=0.016, 95% CI -0.701-0.733). Probiotics showed a beneficial impact on the LAB:coliforms ratio in the subanalysis that included trials that used whole milk (SMD=0.780, 95% CI 0.141-1.418) and monostrain inocula (SMD=0.990, 95% CI 0.340-1.641). The probability of significant effects (probiotic positive effect) in a new study was >0.70 for diarrhea and fecal consistency. Whole milk feeding improved the action of the probiotic effect on the incidence of diarrhea and LAB:coliforms ratio. The probability to find significant effects in the diarrhea frequency and LAB:coliforms ratio was higher (P>0.85) if the new studies were conducted using whole milk to feed calves. This paper defines the guidelines to standardize the experimental designs of future trials. LAB can be used as growth promoters in calves instead of antibiotics to counteract the negative effects of their widespread use.     

RP-HPLC法测定博落回总碱中白屈菜红碱含量的方法研究

建立博落回总碱中白屈菜红碱的RP—HPLC含量测定方法。采用日本资生堂ODS—MG C18液相色谱柱（4．6mm×150mm,5μm）;以乙腈-0．1％磷酸溶液（45：55）（每100mL加十二烷基磺酸钠0．1g）为流动相;检测波长270nm;流速为1．0mL／min。结果显示,白屈菜红碱峰形对称,拖尾因子低于1．5;在0．1μg～0．9μg范围内峰面积与浓度呈良好的线性关系（r=0．9999,n=5）;在80％、100％、120％三种添加水平下的平均回收率分别为97．4％（RSD：0．7％,n=3）、98．6％（RSD=0．1％,n=3）、99．5％（RSD=0．8％,n=3）。利用该法测定博落回总碱中的白屈菜红碱,结果准确度高、精密度好,可作为博落回总碱的质量控制方法。     

A canine model of normovolaemic acute anaemia

The objective was to develop a non-terminal, acute normovolaemic anaemia model in dogs that has minimal effects on patient well-being. Eleven normal Beagle dogs were used. About 20% of the circulating blood volume was removed from the jugular vein 1-3 times per day over a 3-4 day period until a haematocrit (Ht) of 13-17% was obtained. Normovolaemia was maintained by replacing the volume deficit of the red blood cells with Ringer's lactate and re-infusing the plasma. Full blood count and Ht were monitored twice daily. The 13-17% Ht was reached within 3-4 days with the number of phlebotomies ranging from four to seven. The model was primarily developed to determine echocardiographic values as well as Doppler abdominal splanchnic blood flow parameters in anaemic dogs as part of a study that will compare these results to similar studies in babesiosis-induced anaemia. The model may also be useful in the evaluation of the pathophysiology of anaemia in dogs or as a model for anaemia in humans.     

Worm control practices of pig farmers in Greece

Information concerning worm control practices of pig farmers in Greece was collected through a questionnaire survey by visiting farms and interviewing farmers. Questionnaires from 93 farmers were collected. The most commonly treated age groups of pigs were sows (87%), followed by fatteners (59%) and weaners (57%). Most of the farmers treated their sows twice per year (78%). The preferred method for calculating anthelmintic dosages was visual estimation (77%) of an average body weight (97%). Drug administration was by mixing anthelmintics with 1-day ration (97%) in two doses with an interval of 4-5 days (94%). The most commonly used group of anthelmintic was benzimidazoles and probenzimidazoles (37-46%), followed by avermectins (7-14%) and tetrahydropyrimidines (8-14%). Febantel was the most commonly used individual drug (34-40%), followed by ivermectin (7-14%) and pyrantel (9-14%). A small number of pig farmers used more than one group of anthelmintics (1-4%) or individual drug (2-6%) in the same year. The majority of the farmers (60%) used the same anthelmintic group for 3 or more years. When gilts/sows or boars were brought, 55% of the pig farmers did not treat with an anthelmintic. Most of the farmers evaluated the efficacy of an anthelmintic by inspecting the general appearance of the pigs or by inspecting for the presence of worms expelled in the faeces after treatment. Planning of the anthelmintic treatment strategy for most of the farms was the responsibility of veterinarians. However, most of the farmers considered the magnitude of worm infection in their own farm as insignificant.     

Developmental competence of bovine embryos reconstructed by the transfer of somatic cells derived from frozen tissues.

The ability of frozen-thawed fetal skin was examined to generate viable cell lines for nuclear transfer. Fetal skin frozen at -35 degrees C or -80 degrees C in the presence of 5% DMSO were used as tissue explants to generate somatic cells. The resultant confluent cells were then used as donors for nuclear transfer (NT). Of the bovine NT embryos reconstructed from the somatic cells, 78% to 81% showed cleavage, 43% to 48% reached the stage of morula formation and 34% to 35% reached blastocyst formation. There were no significant differences in development (P>0.05) when the NT embryos were compared with those reconstructed from fresh somatic-cell-derived skin tissues (75%, 45%, and 38%, for cleavage, and development to morula and blastocyst stages, respectively). The results indicated that cell lines derived from bovine fetal skin cryopreserved by a simple method could be used as donors in nuclear transfer. The resulting embryos showed similar development in vitro to those reconstructed from unfrozen fetal-skin-derived somatic cells.     

Comparison of bronchodilators in alleviating clinical signs in horses with chronic obstructive pulmonary disease

A representative of each of 3 types of bronchodilators was given iv to horses with clinical signs of chronic obstructive pulmonary disease. We used atropine (0.01 mg/kg of body weight) as an anticholinergic, isoproterenol HCl as a beta-adrenergic, and aminophylline, the soluble salt of theophylline, as a methylxanthine. Response to these drugs was evaluated by measuring maximal change in intrathoracic pressures (delta Ppl) and observing clinical signs. Atropine caused a significant (P less than 0.01) reduction in delta Ppl, compared with that caused by aminophylline. Atropine caused the delta Ppl to decrease an average of 83 +/- 15% toward normal. Clinical improvement was noticed in 87% of the cases. Maximal changes in pleural pressures were reduced by an average of 21 +/- 14 mm of Hg, with 27% returning completely to normal. Isoproterenol helped 77% of horses clinically, and caused delta Ppl to decrease an average of 68 +/- 31% toward normal. The average reduction in delta Ppl was 16 +/- 10 mm of Hg. Aminophylline helped alleviate clinical signs of 50% of the horses and caused delta Ppl to decrease an average of 41 +/- 36% toward normal. The average reduction in delta Ppl was 10 +/- 11 mm of Hg.     

高效液相色谱法测定博落回注射液中血根碱和白屈菜红碱的含量

利用高效液相色谱法测定博落回注射液中血根碱和白屈菜红碱的含量。采用Agilent1100型高效液相色谱仪,色谱柱为AgilentEcpliseXDB—C18（5μm,4．6mm×250mm）,流动相为三乙胺溶液-乙腈（75：25,V／V）,流速1．0mL／min;检测波长270nm;柱温35℃。结果表明：血根碱在0．5～100μg／mL范围内线性关系良好（R2=0．9999）,平均加样回收率（n=3）为96．1％～97．9％,RSD为0．8％～1．8％;白屈菜红碱在0．5～100μg／mL范围内线性关系良好（R2=1）,平均加样回收率（n=3）为96．8％-98．8％,RSD为0．6％～1．0％。本法简单快捷,精密度好,可为制定质控标准提供参考。     

Non-invasive assessment of airway responsiveness in healthy and allergen-sensitised cats by use of barometric whole body plethysmography

This study aimed at determining whether airway responsiveness (AR) tests performed by use of barometric whole body plethysmography (BWBP) were repeatable in cats and to what extent AR was affected by the nebulization protocol used, the age of the animals, the inflammatory status of the airways and prior bronchodilator treatment. Repeatability of AR was tested on two occasions in 30 healthy cats. The concentration of carbachol inducing a 300% increase of the enhanced pause (Penh)--an estimator of airflow limitation--was calculated (C-Penh300) and did not differ significantly between the two tests (0.035+/-0.017% compared to 0.034+/-0.016%) and was significantly and positively correlated. The comparison between rapidly and slowly increasing carbachol concentrations was performed in ten healthy cats and showed a significantly lower C-Penh300 (%) when slowly increasing concentrations were used (0.037+/-0.013% compared to 0.039+/-0.015%, P<0.05). A significant age-related increase of C-Penh300 was evidenced by performing AR tests in 15 healthy cats at 12, 18, 24 and 30 months (12 months: 0.026+/-0.008%, 18 months: 0.031+/-0.009%, 24 months: 0.038+/-0.01%, 30 months: 0.043+/-0.014%, P<0.05). C-Penh300 significantly decreased in 12 Ascaris suum-sensitised cats after allergen exposure (0.026+/-0.016% compared to 0.033+/-0.016%, P<0.05) and was negatively correlated with the granulocyte percentage of bronchoalveolar lavage fluid (r=-0.36, P<0.01). Compared with a placebo inhalation, pre-treatment with inhaled salbutamol significantly increased C-Penh300 in four healthy cats (0.093+/-0.021% compared to 0.036+/-0.004%, P<0.05). This study provides evidence that AR determination by use of BWBP is promising as non-invasive indicator of lower airway inflammation or for monitoring response to bronchodilator treatment.     

Pulse Oximetry in Horses

The clinical usefulness of two pulse oximeters was evaluated at two probe sites in nine anesthetized horses. The hemoglobin saturation determined by the pulse oximeters (SaOx) was compared with the hemoglobin saturation calculated from the measured arterial oxygen tension (SaO2). The mean and standard deviation (SD) were calculated from the differences in saturation measurements, over the saturation range of 80% to 100%, for each oximeter used at the tongue probe site and for one oximeter used at the ear. The oximeter results tended to underestimate the SaO2 with mean differences of -3.7% on the tongue and -6.0% on the ear. The limits of agreement were defined as the mean difference +/- 2 SD. Each oximeter used at the tongue produced limits of agreement of +1% to -8%, which meant that 95% of the SaOx values were 1 percentage point above or 8 percentage points below the SaO2. The variability of the differences and limits of agreement were larger when the ear was used as the probe site and at saturations less than 80%. Although both oximeters tended to underestimate the SaO2, they appeared to be clinically useful in detecting changes in arterial hemoglobin saturation.     

牛传染性鼻气管炎病毒单克隆抗体的制备及阻断ELISA方法的建立

为建立检测牛传染性鼻气管炎病毒(IBRV)血清抗体的阻断ELISA方法,本研究以经蔗糖密度梯度离心法纯化的IBRV作为免疫原制备1株单克隆抗体(MAb),命名为cp-1-1。经间接ELISA、IFA和western blot鉴定,该MAb与IBRV呈阳性反应,与牛病毒性腹泻病毒(BVDV)及牛副流感病毒3型(BPIV3)呈阴性反应,具有较强的特异性。质谱分析结果显示MAb cp-1-1识别的表位位于IBRV VP8蛋白。以纯化的IBRV作为包被抗原、MAb cp-1-1作为检测抗体,建立检测IBRV血清抗体的阻断ELISA方法。该检测方法的抗原包被量为0.89μg/孔,样品稀释度为12,检测抗体MAb量为1.3μg/孔,二抗稀释度为15000。利用50份IBRV抗体呈弱阳性的牛血清(中和抗体效价为14~116)作为标准参考血清,确定该检测方法的阻断率Cut Off值为52.06%,即阻断率高于52.06%时判为阳性,低于52.06%时判为阴性。阻断ELISA方法特异性试验显示仅IBRV阳性血清检测为阳性,而BVDV、BPIV3、牛腺病毒3型(BADV-3)和O型口蹄疫病毒(O-FMDV)阳性牛血清均检测为阴性,表明该方法具有较强的特异性;该方法可检测的最低中和抗体效价为14,与病毒中和试验的敏感性一致,表明该方法具有较高的敏感性;重复性试验显示该方法批内、批间变异系数均小于10%,显示较好的重复性。对130份现地牛血清检测结果显示,该方法与病毒中和试验的符合率为98.46%。用该方法对某牛场接种IBRV灭活疫苗的牛血清进行检测,抗体阳性率为99.51%(205/206)。另外,采用该方法对我国8个省(市、自治区)的801份牛血清进行检测,IBRV的抗体阳性率为41.6%(333/801)。本研究建立的阻断ELISA方法可以用于IBRV疫苗免疫监测和血清流行病学调查,为我国IBR的防控提供技术支持。     

玉米穗腐病样品中黄色镰孢的分离鉴定及rDNA-ITS序列分析

为了明确甘肃玉米穗腐病的病原菌,于2009年9月在甘肃省四大生态区采集玉米穗腐病样品,以组织分离法进行病原物的分离培养,对分离得到的镰孢菌菌落进行纯化和单孢分离后,以形态学为基础,参照Leisle分类系统进行鉴定。结果表明,分离到271株镰孢菌菌株中有105株经形态学鉴定为黄色镰孢,占分离镰孢菌的38.7%。分析发现黄色镰孢种群数量随采样区而异,陇东和陇南地区采集的样品中分离频率分别为52.0%,55.4%,本研究充分证明了黄色镰孢是甘肃省陇东和陇南地区玉米穗腐病的优势病原菌。按照柯赫氏法则用混合菌株接种法对沈单16和金穗96832进行致病性测定,证实了黄色镰孢对玉米果穗的致病性。随机选取3株黄色镰孢菌株进行rDNA-ITS基因序列分析,将PCR产物回收测序后在GenBank 上比对,菌株3-1-1与GenBank 中登记的黄色镰孢菌株K1004和K216、6-4-1和21-2-1与黄色镰孢菌株CBS122.73亲缘关系最近,同源性达99%。利用DNAStar 软件绘制其系统发育树状图,结果表明,菌株3-1-1与K1004和K216、菌株6-4-1和21-2-1与CBS122.73位于系统发育树的同一分支,聚为一类,与形态学的鉴定结果一致。     

"乳源康"对奶牛乳房炎的疗效研究

为验证"乳源康"对奶牛乳房炎的疗效,以青、链霉素为对照药,乳房灌注给药,在奶牛场试用3年,"乳源康"治疗组共收治各类型临床乳房炎病牛1 638头次,2 203个乳区,治愈率为93.3%,总有效率为96.7%,平均疗程4.5 d.青、链霉素对照组共收治各类型临床乳房炎病牛276头次,359个乳区,治愈率为56.3%,总有效率为77.4%,平均疗程12 d;用"乳源康"治疗隐性乳房炎4 031例,用药1次～2次临床治愈率为100%;通过对56个患病乳区"乳源康"用药前后的细菌削减状况的检查,细菌转阴率为71.74%.因此,用"乳源康"治疗奶牛乳房炎安全、高效.     

Morphological features of spermatozoa of swamp buffalo AI bulls in Thailand

The appearance and incidence of sperm abnormalities was studied in 115 ejaculates, collected periodically over 1 year covering all seasons from five mature, healthy swamp buffalo (Bubalus bubalis) bulls reared under tropical conditions and serving as the current source of semen for artificial insemination (AI) in Thailand. Light microscopy of stained smears was used to investigate sperm head shape morphology, while unstained wet smears were used to examine other sperm abnormalities. The most commonly found morphological aberrations were pear-shaped spermatozoa, knobbed acrosomes, proximal cytoplasmic droplets, simple bent tails and coiled tails under the head, whose ultrastructure (scanning electron microscopy) corresponded to what has been found in other species of bovidae, including varieties of buffalo. The mean prevalence (as least squares mean +/- SEM) of sperm abnormalities was low (below 15%), corresponding to healthy spermiograms. The younger bulls (<10 years old, n = 3) had less abnormalities than the older ones (10.1 +/- 0.6% versus 14.1 +/- 0.8%, P < 0.001, n = 2), including abnormalities of sperm head shape (1.1 +/- 0.3% versus 3.6 +/- 0.3, P < 0.001), acrosome defects with knobbed acrosomes (1.1 +/- 0.2% versus 1.2 +/- 0.3%, P < 0.001), spermatozoa with proximal cytoplasmic droplets (2.7 +/- 0.1% versus 1.4 +/- 0.2%, P < 0.001), defective mid-pieces (0.2 +/- 0.1% versus 0.3 +/- 0.1%) and abnormal sperm tails (3.1 +/- 0.3% versus 5.7 +/- 0.4%, P < 0.001). The within-bull effect of the year solely affected the incidence of pear-shaped spermatozoa while the incidences of abnormal contour, variable size of sperm head shapes, abnormal mid-piece and simple bent tail among bulls were affected by ejaculate (week of collection). Interaction between age and ejaculate affected only the prevalence of spermatozoa with proximal cytoplasmic droplets. In conclusion, the types of defects encountered were similar to those found in other bovidae, with a very low prevalence over the year the AI sires were followed through.     

气相色谱法测定苦参苍术口服液中三氯甲烷残留量

建立了苦参苍术口服液中三氯甲烷残留量的气相色谱检测方法,采用DB—FFAP毛细管柱,柱温为程序升温,氢火焰离子化检测器（FID）,检测器温度为250℃,进样口温度为220oC,载气为氮气。试验结果三氯甲烷在6．0464～120．928μg／mL浓度范围内呈良好的线性关系（r=0．99995）,加样回收实验平均回收率为98．01％（RSD=1．6％）。本方法简便、快速、准确、重现性好,可作为苦参苍术口服液中三氯甲烷残留量的检测方法。     

Pharmacokinetics of tramadol in horses after intravenous, intramuscular and oral administration

Tramadol is a centrally acting analgesic drug that has been used clinically for the last two decades to treat moderate to moderately severe pain in humans. The present study investigated tramadol administration in horses by intravenous, intramuscular, oral as immediate-release and oral as sustained-release dosage-form routes. Seven horses were used in a four-way crossover study design in which racemic tramadol was administered at 2 mg/kg by each route of administration. Altogether, 23 blood samples were collected between 0 and 2880 min. The concentration of tramadol and its M1 metabolite were determined in the obtained plasma samples by use of an LC/MS/MS method and were used for pharmacokinetic calculations. Tramadol clearance, apparent volume of distribution at steady-state, mean residence time (MRT) and half-life after intravenous administration were 26+/-3 mL/min/kg, 2.17+/-0.52 L/kg, 83+/-10 min, and 82+/-10 min, respectively. The MRT and half-life after intramuscular administration were 155+/-23 and 92+/-14 min. The mean absorption time was 72+/-22 min and the bioavailability 111+/-39%. Tramadol was poorly absorbed after oral administration and only 3% of the administered dose was found in systemic circulation. The fate of the tramadol M1 metabolite was also investigated. M1 appeared to be a minor metabolite in horses, which could hardly be detected in plasma samples. The poor bioavailability after oral administration and the short half-life of tramadol may restrict its usefulness in clinical applications.     

Coelomic implantation of satellite transmitters in the bar-tailed godwit (Limosa lapponica) and the bristle-thighed curlew (Numenius tahitiensis) using propofol, bupivacaine, and lidocaine

Intravenous propofol was used as a general anesthetic with a 2:1 (mg:mg) adjunctive mixture of lidocaine and bupivacaine as local anesthetics infiltrated into the surgical sites for implantation of satellite transmitters into the right abdominal air sac of 39 female and 4 male bar-tailed godwits (Limosa lapponica baueri and Limosa lapponica menzbeiri and 11 female and 12 male bristle-thighed curlews (Numenius tahitiensis). The birds were captured on nesting grounds in Alaska, USA, and on overwintering areas in New Zealand and Australia from 2005 through 2008. As it was developed, the mass of the transmitter used changed yearly from a low of 22.4 +/- 0.2 g to a high of 27.1 +/- 0.2 g and weighed 25.1 +/- 0.2 g in the final year. The mean load ratios ranged from 5.2% to 7.7% for godwits and from 5.7% to 7.5% for curlews and exceeded 5% for all years, locations, and genders of both species. The maximum load ratio was 8.3% for a female bar-tailed godwit implanted in Australia in 2008. Three godwits and no curlews died during surgery. Most birds were hyperthermic upon induction but improved during surgery. Two godwits (one in New Zealand and one in Australia) could not stand upon release, likely due to capture myopathy. These birds failed to respond to treatment and were euthanized. The implanted transmitters were used to follow godwits through their southern and northern migrations, and curlews were followed on their southern migration.     

赛里木拉丝酸奶中优势菌株发酵乳对血管紧张素转化酶的抑制作用

利用改进的紫外分光光度法,测定从新疆赛里木拉丝酸奶中分离出的瑞士乳杆菌mb2-1、嗜热链球菌mb5-1以及马克斯克鲁维酵母Y6单菌和混菌发酵脱脂乳对血管紧张素转化酶抑制率。结果表明:瑞士乳杆菌mb2-1、嗜热链球菌mb5-1单菌发酵对血管紧张素转化酶抑制率48h达到最高,分别为70.75%、47.17%;与马克斯克鲁维酵母Y6混合发酵48h,对血管紧张素转化酶抑制率分别为75.68%和44.85%。     

Development of enzyme-linked immunosorbent assay with nucleoprotein as antigen for detection of antibodies to avian influenza virus

During the avian influenza outbreak of 2003-04 in Southeast Asia, two avian influenza viruses (AIV), one of H5N1 subtype and the other H9N2 subtype, were isolated and identified from local farms. The nudeoprotein (NP) gene of the H5N1 AI isolate was cloned, and the segment encoding amino acid 47-384, which covers its major antigenic domains, was subcloned and expressed in E. coli. Subsequently, the NP (47-384) expression product was purified and used as the diagnostic antigen to develop a NP-based type-specific indirect enzyme-linked immunosorbent assay (ELISA) for detecting antibodies to AI from chicken sera. The ELISA is shown to be specific for AIV and does not cross-react with chicken sera that has antibodies to other avian viruses. The NP(47-384)-ELISA was compared with a hemagglutination inhibition test and a commercial AIV ELISA kit in evaluating 150 sera samples from experimentally AIV-infected or vaccinated specific-pathogen-free (SPF) chickens. Our NP(47-384)-ELISA was more sensitive than the two tests and showed an 82% agreement ratio with the HI test and an 80.67% agreement ratio with the commercial kit. The NP(47-384)-ELISA and the commercial AIV ELISA were used to evaluate 448 field sera samples from diseased chickens or vaccinated chickens during the 2003-04 AI outbreak in China. The two ELISA tests had a 95% agreement ratio. We conclude that the NP(47-384)-ELISA developed in our laboratory was specific and sensitive and it has great application potential in China's long-term prevention and control of AI.     

Seroprevalence and risk factors associated to Bovine Herpesvirus 1 (BHV-1) infection in non-vaccinated dairy and dual purpose cattle herds in Ecuador

A cross-sectional study was carried out to determine the seroprevalence and risk factors associated to Bovine Herpesvirus 1 (BHV-1) infection in non-vaccinated dairy and dual purpose cattle herds from Ecuador. A total of 2367 serum samples from 346 herds were collected from June 2008 through February 2009. A questionnaire, which included variables related to cattle, health, management measures and environment was filled out in each herd. A commercial indirect ELISA test was used to determine the seropositivity against BHV-1. Generalized Estimating Equations (GEE) model was used to determine risk factors at individual level, including herd as random effect. The individual seroprevalence to BHV-1 in Ecuador was 43.2% (1023/2367; CI(??%): 41.2-45.2%). The herd prevalence was 82.1%; (284/346; CI(??%): 78.1-86.1%) and the intra-herd prevalence ranged from 12.5 to 100% (mean=64.1%). The GEE model showed that animal age (>4 years) (OR: 1.44; CI(??%): 1.18-1.75), BRSV infection (OR: 1.45; CI(??%): 1.09-1.92), altitude over the sea level (≤ 1800 m) (OR: 2.97; CI(??%): 2.1-4.22) and average slope (> 11%) (OR: 1.45; CI(??%): 1.07-1.95) are risk factors associated with BHV-1 infection, while a good cleaning of the facilities (OR: 0.66; CI(??%): 0.44-0.99) was shown to be a protective factor.