Rapid PCR detection of Salmonella in horse faecal samples

A rapid polymerase chain reaction (PCR) assay was developed for detecting Salmonella in faeces of horses and assessed on samples from horses admitted to a veterinary hospital. Direct detection was achieved by amplification of part of ompC after extraction of DNA from faeces using a spin column method to reduce the amount of inhibitory substances in samples. An internal positive control was included to detect false negative results. While the sensitivity of the PCR assay was less than culture when assessed on faeces inoculated with Salmonella, its sensitivity on faecal samples obtained from horses was much greater than culture. Salmonella DNA was detected in 40% of faecal samples using the PCR assay while Salmonella were cultured from only 2% of the samples. The PCR assay has potential for use in either routine diagnosis or for detection of the carrier status in animals.     

Detection of Mycoplasma hyopneumoniae in lung and nasal swab samples from pigs by nested PCR and culture methods

We examined nasal swab and lung homogenate samples collected from pigs experimentally and naturally infected with Mycoplasma hyopneumoniae for the detection of M. hyopneumoniae by the nested PCR (nPCR) and culture methods. In the 23 experimentally infected pigs, M. hyopneumoniae was commonly detected in nasal swabs by the nPCR and culture methods at 4 weeks after inoculation, and there was a significant correlation (P<0.01) between the titers of viable organisms in nasal swabs and in lung homogenates in the experimentally inoculated pigs. In the naturally infected pigs, on the other hand, discrepancies in detection were found between nasal swab and lung homogenate samples in 17 of 36 cases, although the presence of gross lung lesions correlated relatively well with the detection of organisms from the samples. Our results indicated that the diagnosis of mycoplasmal pneumonia by nPCR in individual pigs with nasal swabs is reliable under these experimental conditions. At present, nPCR with nasal swabs should only be used for monitoring the disease status at the herd level under field conditions.     

Different feed withdrawal times before slaughter influence caecal fermentation and faecal Salmonella shedding in pigs

The effects of different pre-slaughter feed withdrawal times (FWT) on the gastrointestinal tract (GIT) weight and the gut environment of pigs and Salmonella shedding were investigated. Trial 1 evaluated the effects under experimental conditions (feed withdrawal for 18, 30 and 36 h) and trial 2 under commercial conditions (15 and 30 h). In trial 1, the GIT weight tended to decrease (P=0.07), the caecal pH increased (P<0.0001), short-chain fatty acids (SCFA) decreased (P<0.001) and percentage of branched-chain fatty acids (BCFA) increased as FWT increased. Similar results were observed in trial 2, but Enterobacteriaceae numbers and Salmonella positive pigs tended to increase whereas lactobacilli decreased (P<0.0005) as FWT increased. The increase in FWT involved changes in the gut microbial ecosystem that could be associated with the trend of increased caecal Enterobacteriaceae and Salmonella in faeces, and may represent a higher risk of carcass contamination in cases of laceration of viscera.     

Comparison of five culture methods for Salmonella isolation from swine fecal samples of known infection status

The current study was conducted to evaluate 5 bacteriologic culture methods (methods 1, 2, 3, 4, and 5) for recovery of Salmonella enterica from swine feces, both for sensitivity of detection (ability to recover Salmonella from a positive sample) and for specificity (not to inadvertently identify an organism as a Salmonella species in a negative sample). Fifty-six negative samples and 46 positive samples were processed using each of the 5 methods, which differed primarily in the combinations of enrichment media used. All negative samples were negative for Salmonella when cultured by all 5 methods (100% specificity). Two of the methods (methods 1 and 4) resulted in the recovery of significantly less (P < 0.05) Salmonella when compared with the remaining 3 methods (methods 2, 3, and 5). No one method was successful in recovering Salmonella from all positive samples, although recovery with method 2 was statistically similar to the total number of positive samples analyzed (42 vs. 46 Salmonella-positive samples, P > 0.05). This study shows that culture methods differ significantly in their performance regarding the isolation of Salmonella from swine fecal samples.     

Salmonella (sero)types and their resistance patterns in pig faecal and post-mortem samples.

The purpose of this survey was to take stock of porcine Salmonella isolates derived from faecal and post-mortem samples over a 4-year period. Salmonella was isolated by direct inoculation on BGA(NO)-plates (faeces, intestinal content) or sheep blood agar (organs). Antimicrobial susceptibility was tested by the agar diffusion method. Salmonella was isolated in 4.2% of all porcine submissions received at the Animal Health Service. A total of 1305 salmonellae were isolated from a total of 1279 submissions from 1008 different herds. Salmonella Typhimurium was the most frequently isolated serotype (88%), and Salmonella Typhimurium DT104 was the most frequently isolated phagetype (17.2% of Salmonella Typhimurium). Resistance to antimicrobials occurred in 47.3% of all isolates, mainly those of the multiresistant phagetype DT104. Other pathogens were isolated in more than 50% of the submissions. In cases of clinical diarrhoea, multiple pathogens may be involved and therapy and preventive measures should be adjusted accordingly.     

动物粪便沙门菌PCR检测技术的研究

为了建立一种动物粪便样品中快速、特异、敏感的沙门菌检测方法,根据沙门菌肠毒素stn基因设计一对引物,对不同血清型的沙门菌和非沙门菌进行PCR检测,并对该PCR方法进行反应的灵敏度、模拟粪便样品的最低检出限测定及粪便样品的检测。运用该方法对250份粪便样品进行检测,同时用传统方法进行验证。结果表明,设计的引物特异性好,能专一性扩增出约260 bp条带;该引物灵敏度高,能进行有效检测的核酸最低起始量为43.85 pg,纯菌检测灵敏度达1 cfu/mL。接种沙门菌到粪便样品中,当粪便样本中菌量达103cfu/mL以上时,不需要增菌,沙门菌可立即检出,增菌后检出限可以达到1 cfu/mL。PCR检测250份样品共检出18份阳性,同时传统细菌培养方法证明结果正确。该方法可用于粪便样品的检测。     

Detection of Mycoplasma synoviae in poultry environment samples by culture and polymerase chain reaction

Successful detection of Mycoplasma synoviae (MS) by culture and PCR from samples collected in the environment of experimentally infected chickens and turkeys, or under field conditions, is described. Results showed that in the experimental infection, 10/96 and 46/96 samples of food, drinking water, feathers, droppings or dust were positive by culture and Mycoplasma-PCR. In field conditions, the number of positive results for environmental samples were respectively 7/28 and 17/28. These observations highlight the high disseminating capacities of this mycoplasma and show the usefulness of the PCR method for epidemiological studies.     

Evaluation of culture methods for rapid screening of swine faecal samples for Yersinia enterocolitica O:3/biotype 4.

In two studies, seven different culture protocols were compared to test naturally contaminated faecal samples from pigs for isolation of Y. enterocolitica serotype O:3/biotype 4 (n = 70 and n = 79). Four of the protocols were based on the Nordic Committee on Food Analysis (NMKL, protocols), while three protocols were based on a rapid and selective method (here called ITC protocols). The protocols differed mainly in time of pre-enrichment (1, 10 and 24 d) and enrichment (2, 10, 24 d) and the type of selective enrichment media (ITC vs. MRB). The sensitivity of the rapid ITC protocol (24% and 9%) was comparable with the lengthy NMKL-protocols (16% and 11%), while the results of direct plating after 3 h (4%) and the extended enrichment in ITC-broth (4%) were very low. In addition, there was a marked reduction in the number of false positive plates in the short selective protocol (62% vs. 12%). The results indicate possibilities of shortening the culture methods by replacing most of the biochemical tests with an agglutination test based on a monoclonal antibody.     

Growth and parameters of microflora in intestinal and faecal samples of piglets due to application of a phytogenic feed additive

A commercial phytogenic feed additive (PFA), containing the fructopolysaccharide inulin, an essential oil mix (carvacrol, thymol), chestnut meal (tannins) and cellulose powder as carrier substance, was examined for effects on growth and faecal and intestinal microflora of piglets. Two experiments (35 days) were conducted, each with 40 male castrated weaned piglets. In experiment 1, graded levels of the PFA were supplied (A1: control; B1: 0.05% PFA; C1: 0.1% PFA; D1: 0.15% PFA) in diets based on wheat, barley, soybean meal and fish meal with lysine as the limiting amino acid. In experiment 2, a similar diet with 0.1% of the PFA (A2: control; B2: 0.1% PFA; C2: +0.35% lysine; D2: 0.1% PFA + 0.35% lysine) and lysine supplementation was utilized. During experiment 1, no significant effect of the PFA on growth, feed intake and feed conversion rate was observed (p > 0.05). Lysine supplementation in experiment 2 improved growth performance significantly, but no significant effect of the PFA was detected. Microbial counts in faeces (aerobes, Gram negatives, anaerobes and lactobacilli) during the first and fifth week did not indicate any significant PFA effect (p > 0.05). In addition, microflora in intestinal samples was not significantly modified by supplementing the PFA (p > 0.05). Lysine supplementation indicated lysine as limiting amino acid in the basal diet, but did not influence the microbial counts in faeces and small intestine respectively.     

Optimization of a culture technique for the isolation of Listeria monocytogenes from faecal samples

A culture technique employing cold enrichment at 4 degrees C followed by selective enrichment and plating at higher temperatures (30 degrees C) was used to isolate Listeria monocytogenes from faecal samples. The samples were held at 4 degrees C for 15 weeks and cultured weekly to assess the sensitivity of the culture after cold storage for different lengths of time. No media, Listeria selective enrichment broth (LSEB), nutrient broth (NB) and saline were used as cold storage medium. Cold storage increased the frequency of Listeria positive samples. The sensitivity of the culture for Listeria spp. and L. monocytogenes was 72 and 94%, and 56 and 61% after third and seventh week of cold storage, respectively. When the results of third and seventh week of cold storage were combined, the sensitivity was 100% for Listeria spp. and 94% for L. monocytogenes. LSEB and NB as storage medium increased Listeria positive samples after the first week of cold storage but did not maintain the increase thereafter while saline had an adverse effect on the growth of the bacteria. However, samples held in no media in a pilot study involving monthly sampling of a herd revealed better results. Detection limit of the culture media was also investigated. The lowest concentration detected by culture media was 3.17 organisms/ml. This was seven organisms/g for known Listeria positive sample. The faecal samples spiked with 10-fold dilutions of L. monocytogenes and held at 4 degrees C revealed that the sample spiked with 3.17 x 10-1 cfu/ml organisms resulted in growth after the second week of cold storage. The results suggest that the culture technique employing cold enrichment followed by selective enrichment and plating is more sensitive, the storage of faecal samples in no media when compared with the samples in storage medium, LSEB, NB and saline, during cold enrichment is a better application and culture of faeces, immediately after collection, at third and seventh week of cold enrichment produce more satisfactory results.     

湖北省饲料及饲料添加剂抽检分析

饲料及饲料添加剂产品的优劣直接关系到畜牧业产品的质量 ,进而影响人们的健康安全 ,同时也将影响我国饲料及饲料添加剂产品和畜牧业产品的出口。在农业部统一安排下 ,我省于 2 0 0 2年分三期对湖北省境内的饲料及饲料添加剂生产厂家、销售市场及养殖企业进行了认真的抽样检测。现将此次抽样检测资料整理如下 ,为主管部门、生产经营及养殖企业提供参考信息。全省此次共抽检企业 85 5个次 ,合格企业689个次 ,合格率 80 5 8% ;抽检产品 1 642个样品 ,合格率 85 5 7%。生产、经营、养殖企业的产品合格率分别为 84 69%、68 99%和 99 2 0 %。不…     

The effect of dietary garlic supplementation on body weight gain,feed intake,feed conversion efficiency,faecal score,faecal coliform count and feeding cost in crossbred dairy calves

Thirty-six crossbred calves (Holstein cross) of 5 days of age were used to study the effect of garlic extract feeding on their performance up to the age of 2 months (pre-ruminant stage). They were randomly allotted into treatment and control groups (18 numbers in each group). Performance was evaluated by measuring average body weight (BW) gain, feed intake (dry matter (DM), total digestible nutrient (TDN) and crude protein (CP)), feed conversion efficiency (FCE; DM, TDN and CP), faecal score, faecal coliform count and feeding cost. Diets were the same for the both groups. In addition, treatment group received garlic extract supplementation at 250 mg/kg BW per day per calf. Body weight measured weekly, feed intake measured twice daily, proximate analysis of feeds and fodders analysed weekly, faecal scores monitored daily and faecal coliform count done weekly. There was significant increase in average body weight gain, feed intake and FCE and significant decrease in severity of scours as measured by faecal score and faecal coliform count in the treatment group compared to the control group (P < 0.01). Feed cost per kilogramme BW gain was significantly lower in the treatment group compared to control group (P < 0.01). The results suggest that garlic extract can be supplemented to the calves for better performance.     

猪链球菌2型荧光PCR检测方法与常规检测方法的比较

为验证猪链球菌2型荧光PCR检测方法对临床样品检测的敏感性和适用性以及该方法所拥有的独特优点,分别用荧光PCR法、常规PCR法和细菌分离法对人工感染猪链球菌2型的小鼠肝脏和发病猪的心、肝、脾、肾等实质器官和血液、喉拭子进行抗原检测。结果显示,荧光PCR法检出率为70.8%,明显高于普通PCR法（检出率为20.8%）,也高于常规细菌分离法（检出率为45.8%）。由于临床样品常常会被其他细菌污染,细菌分离法很难准确分离到链球菌。但荧光PCR法不受其他细菌污染的影响,对实验室培养的猪链球菌2型菌液,该方法检测滴度可达10-6/0.1mL（42～52 CFU/0.1 mL）,而普通PCR方法检测滴度仅为10-4。     

Detection and identification of Salmonella Typhimurium in bovine diarrhoeic fecal samples by immunomagnetic separation and multiplex PCR assay

The aim of this study was to use the immunomagnetic separation (IMS) test plus a multiplex polymerase chain reaction (m-PCR) assay to detect Salmonella at genus level and also for the identification of Salmonella enterica serovar Typhimurium in bovine diarrhoeic fecal samples. In all, 400 bovine diarrhoeic fecal specimens were examined by conventional bacterial culture, IMS, and m-PCR. For m-PCR assay, four set primers were selected: 139-141, specific for inv-A gene of Salmonella spp and the RfbJ, FliC and FljB, specific for the rfbJ, FliC and fljB genes of Salmonella Typhimurium or other Salmonella serovars with similar antigenic properties. Thirty-three (8.25%) out of the 400 fecal samples were culture positive for Salmonella serovars. Of these, 66.7% (22 of 33) were Salmonella enterica serovar Typhimurium, and 9.1% (three of 33) were serovar Dublin. In the IMS + m-PCR, four amplified product (663, 526, 284 and 183 bp) were found in all specimens that had serovar Typhimurium (4,5,12:i:1,2), they corresponded, respectively, to the rfbJ, fljB, inv-A and Flic genes of this serovar. In serovar Dublin (1,9,12:g,p:-), Georgia (6,7:b:e,n,z(15)) and, Enteritidis (1,9,12;g,m:-) only one PCR product (284 bp) was amplified from the inv-A gene. In serovars Augustenborg (6,7:i:1,2) and Lindenburg (6,8:i:1,2) three positive bands (526, 284 and 183 bp) were amplified corresponding to the fljB, inv-A and Flic genes, respectively. In serovar Virchow (6,7:r:1,2) two amplified products (284 and 526 bp) from the inv-A and FliC genes were observed. In serovar Gloucster (1,4,12(27):i:1,w) three fragments (183, 284 and 663) from the FliC, inv-A and, rfbJ genes respectively, were observed. In the positive control as expected, four PCR products were amplified corresponding to the FliC, inv-A, fljB and rfbJ genes, respectively. In conclusion, the results of this study showed that detection of Salmonella at genus level with universal ST139-141 primers and identification of Salmonella Typhimurium by using specific primers of O4, H(2):1, 2 and H(1) antigens can potentially permit to more readily evaluate fecal and other types of samples for the presence of these organisms. Compared to bacteriological culture the combination of IMS and m-PCR resulted a faster method for the detection and identification of Salmonella at genus and serovar level by using of universal and specific primers.     

Detection of rhodococcus equi by microbiological culture and by polymerase chain reaction in samples of tracheobronchial secretions of foals

The goal of the present study was to investigate whether new PCR-methods would improve diagnostic of R. equi. In a first step, sensitivity and specificity of the PCR-methods in respect to the"gold standard" microbiological culture were determined. Secondly, sensitivity and specificity of both microbiological methods were evaluated in respect to the clinical diagnosis. The tracheobronchial secretions of 48 foals with pulmonary abscesses and of 37 healthy foals were evaluated by bacteriological culture as well as by four PCR-methods: aceA-, ideR-, vapA- and VP-PCR. In respect to the"gold standard" microbiological culture, the sensitivity of most PCR methods lay between 63.9 and 69.4 % except the vapA-PCR (27.8 %).The specificity of all PCR methods in this comparison was between 98 to 100 %. In this analysis, clinical diagnosis had a low sensitivity (66.7 %) and a low specificity (51.0 %). In respect to the clinical diagnosis, microbiological culture sensitivity was 50.0 % and specificity 67.7 9%. In this analysis, sensitivity rates of aceA-, ideR and VP-PCR methods lay between 33.3 and 37.5 %, sensitivity of the vapA-PCR was lower (10.4 %).The specificity of all PCR methods ranged from 78.4 to 86.5 %. In conclusion, these results show that the diagnostic potential of the microbiological methods"Culture" and "PCR" is different and that for the diagnosis of R. equi-pneumonia in foals the combination of microbiological culture with PCR should be used for examination of samples of the airways of foals.     

Comparative sensitivity of various faecal culture methods and ELISA in dairy cattle herds with endemic Johne's disease

In three New South Wales dairy cattle herds with endemic Johne's disease, prevalence rates by faecal culture were determined to be 12, 18 and 22%, respectively. Whole herd faecal culture was shown to detect markedly more infected cattle than whole herd testing by the EMAI absorbed ELISA, particularly in the two herds with greatest prevalence. In the three study herds, five methods for whole herd faecal culture were compared in each. These included two methods based on primary culture on Herrold's egg yolk medium with mycobactin J (HEYM): (1) conventional decontamination with sedimentation and primary culture on HEYM; (2) Whitlock decontamination and culture on HEYM. The remaining three methods were based on radiometric (BACTEC) culture: (3) decontamination and filtration to BACTEC medium; (4) modified Whitlock decontamination to BACTEC medium and (5) Whitlock decontamination to BACTEC medium. For BACTEC cultures, two methods were compared as confirmatory tests for Mycobacterium paratuberculosis: mycobactin dependence on conventional subculture to HEYM and IS900 PCR analysis of radiometric media.Among 179 cattle tested simultaneously by all five culture methods, 38 cattle were confirmed to be shedding M. paratuberculosis. In identifying shedder cattle, method 5 was the most sensitive, followed by methods 2, 4, 1, and 3 was the least sensitive. The number of BACTEC cultures confirmed by mycobactin dependence or PCR was similar.