Changes in caecal microbiota and mucosal morphology of weaned pigs

An experiment was designed to monitor the changes in caecal microbiota associated with early weaning. Twelve piglets (20+/-2 days) from six different litters were selected from a commercial source. For the two experimental groups, one animal from each litter was weaned onto a post-weaning diet (W) and the other remained with the sow (S). After 1 week, animals were sacrificed and caecal samples taken. Microbial counts for total bacteria, enterobacteria and lactobacilli populations were determined by quantitative PCR using SYBR Green dye. Microbial profiles were assessed by terminal restriction fragment length polymorphism (t-RFLP). Weaning promoted an increase in the enterobacteria:lactobacilli ratio (0.27 versus 1.67 log/log 16S rRNA gene copy number, P=0.05). Total bacteria and richness of the caecal microbial ecosystem (number of peaks) were similar in both experimental groups (49.3 for S and 53.4 for W, respectively, P=0.22), although the band patterns were clearly grouped in two different clusters by dendogram analysis. Weaning was also associated with a decrease in crypt density, an increase in mytotic index and a decrease in the number of goblet cells. A reduced immunological response was also observed and was manifested by an increase in intraepithelial lymphocytes and lymphocyte density in the lamina propria. Weaning appears to be critical in the establishment of the caecal microbiota in pigs with important changes, particularly in microbial groups and in caecal mucosal architecture.     

Detection of equine herpesvirus-1 in nasal swabs of horses by quantitative real-time PCR

Background: Early identification of inhalation-transmitted equine herpesvirus type 1 (EHV-1) infections has been facilitated by the availability of a number of real-time quantitative PCR (qPCR) tests. A direct comparison between nasal swab qPCR and traditional virus isolation (VI) requires a method for normalizing the qPCR samples and controlling for PCR inhibitors present in some clinical samples.
Objectives: To quantify EHV-1 shedding in viral swabs using an internal control and to compare fast qPCR to VI for the detection of EHV-1 in nasal swabs from horses.
Animals: Fifteen horses experimentally infected with EHV-1.
Methods: Experimental study : Nasal swab samples were collected daily after experimental infection for up to 21 days. VI was performed by conventional methods. The DNA was prepared for qPCR with the addition of a known quantity DNA of Marek's disease virus as an internal control. qPCR was performed.
Results: The qPCR method detected virus up to day 21 after challenge, whereas VI detected virus only to day 5. The median Kaplan-Meier estimates for EHV-1 detection were 12 days for qPCR and 2 days for VI ( P < .0001). When compared with VI, the sensitivity and specificity of qPCR were 97 (95% CI: 86–100) and 27% (95% CI: 20–35).
Conclusions and Clinical Importance: We conclude that fast qPCR of nasal swab samples should be chosen for diagnosis and monitoring of herpesvirus-induced disease in horses. Recommended reference ranges of C _T values are provided as well as justification of a minimum 10-day quarantine period.     

Use of mannanoligosaccharides and zinc chelate as growth promoters and diarrhea preventative in weaning pigs: Effects on microbiota and gut function

The efficacy of a commercial source of mannanoligosacharides (BM), organic zinc (BP), or their combination to enhance performance, gastrointestinal health, and immune response in weaned pigs was evaluated. A total of 128 piglets, weaned at 20 +/- 2 d, were housed in 32 pens. Animals received 1 of 4 dietary treatments: a control diet (CT) to which 0.2% of BM, 80 mg/kg of Zn as BP, or both additives (BMP) were added. The experiment lasted for 5 wk including a prestarter period of 2 wk and a starter period of 3 wk. Body weight was recorded and daily feed intake was calculated. Fecal consistency was monitored for the first 21 d. After 2 wk, 32 animals were killed, digesta samples from the stomach, ileum, and cecum were collected, and pH and the short-chain fatty acid profile were determined. Microbiological counts for enterobacteria and lactobacilli were evaluated using quantitative PCR. Histological parameters in the jejunum and immunoglobulin concentrations in serum and ileal digesta were also measured. Both additives improved G:F during the starter period (0.63, 0.69, 0.67, and 0.68 for CT, BM, BP, and BMP, respectively; P < 0.04). Mean fecal score values for the first 21 d were improved by BM and BP, showing decreased values compared with the CT diet (1.22, 0.89, 0.87, and 1.06 for CT, BM, BP, and BMP, respectively; P = 0.002). The addition of BM decreased enterobacteria counts in the jejunum (9.13, 8.05, 8.87, and 7.89 log 16S rRNA gene copies/g of matter for CT, BM, BP, and BMP, respectively; P = 0.05). Empty ileal weight, defined as the segment including the continuous Peyer's patch, tended (P = 0.08) to increase with BP treatment (8.9, 9.6, 11.9, and 10.3 g/kg of BW for CT, BM, BP, and BMP, respectively). Crypt depths in the jejunum were lower in animals fed the combination of the additives (BPM) compared with those fed the control diet (281 vs. 235; P < 0.03). No significant differences were registered in pH, short-chain fatty acids, or serum and ileal immunoglobulin concentrations. The results suggest that the use of BM or BP can improve the efficiency of gain during the starter period.     

The response of gastrointestinal microbiota to avilamycin, butyrate, and plant extracts in early-weaned pigs

An experiment was designed to evaluate the effects of 3 different additives on the gastrointestinal microbiota of early-weaned pigs. Early-weaned (18 to 22 d; n = 32) pigs (6.0 +/- 0.10 kg of BW) from 8 litters were randomly distributed into 8 pens. Each pen was assigned 1 of 4 dietary treatments: a prestarter or control diet, the control diet with 0.04% avilamycin (AB), with 0.3% sodium butyrate, or with 0.03% plant extract mixture (XT; standardized mixture with 5% (wt/wt) carvacrol extracted from Origanum spp., 3% cinnamaldehyde extracted from Cinnamonum spp., and 2% capsicum oleoresin from Capsicum annum). At the end of the experimental period, 8 pigs per treatment were killed, and samples of their intestinal content were taken. The total bacterial load along the gastrointestinal tract (GIT; stomach, jejunum, cecum, and distal colon) and the lactobacilli and enterobacteria in the jejunum and cecum were measured by quantitative PCR. The total microbial counts along the GIT did not differ among the diets, but there was an increase in the lactobacilli:enterobacteria ratio in the cecum of the piglets on the XT diet (P = 0.003). Restriction fragment length polymorphism of the PCR-amplified V3, V4, and V5 regions of the 16S rDNA gene showed changes in the structure of the microbial community in the jejunum. Dendrograms grouped animals by diets; control with 0.3% sodium butyrate was the treatment that promoted the biggest changes in the microbial ecosystem, followed by AB and then XT. Biodiversity increased when using additives compared with the control diet (P = 0.002). Microbial metabolic activity along the hindgut was studied using the concentration of purine bases and carbohydrase activities. Different patterns for purine bases were observed between diets (diet x intestinal section, P = 0.01). The control diet reached a maximum purine base concentration at the end of the colon, whereas that of the AB diet was reached at the cecum. We could not detect any cellulase or xylanase activities in animals of this age. Appreciable amylase and amylopectinase activities were found, but they did not differ between diets. The results suggest that the effects of avilamycin, butyrate, or the plant extract would not be related to a reduction in the number of total bacteria inhabiting different sections of the GIT but rather to changes in the ecological structure and metabolic activity of the microbial community.     

Detection of Actinobacillus pleuropneumoniae in pigs by real-time quantitative PCR for the apxIVA gene

A real-time quantitative PCR (qPCR) for detection of the apxIVA gene of Actinobacillus pleuropneumoniae was validated using pure cultures of A. pleuropneumoniae and tonsillar and nasal swabs from experimentally inoculated Caesarean-derived/colostrum-deprived piglets and naturally infected conventional pigs. The analytical sensitivity was 5colony forming units/reaction. In comparison with selective bacterial examination using tonsillar samples from inoculated animals, the diagnostic sensitivity of the qPCR was 0.98 and the diagnostic specificity was 1.0. The qPCR showed consistent results in repeatedly sampled conventional pigs. Tonsillar brush samples and apxIVA qPCR analysis may be useful for further epidemiological studies and monitoring for A. pleuropneumoniae.     

Adaptation of gut microbiota to corn physical structure and different types of dietary fibre

An experiment was designed to study the effect of corn physical structure and different types of dietary fibre on pig gut microbiota during growing. Ninety-six animals (15 ± 2.2 kg) were assigned to four different diets: a control diet (CT), a diet with coarse ground corn (CC), and other two with 8% of sugar beet pulp (BP) or 10% of wheat bran (WB). Thirty-two animals (8 per diet) were sacrificed on days 7, 21 and 42 to measure microbial activity in digesta. Other 8 pigs were sacrificed on day 0 before receiving the diets. Enterobacteria and lactobacilli population in colon were quantified by qPCR. With all diets enterobacteria diminished with time and were lower than lactobacilli. Expressed as lactobacilli:enterobacteria ratio we found differences between diets at day 7 and 21 that had disappeared at day 42. Purine bases (PB) concentration indicated an adaptation of the microbiota with time which was reflected in an increasing content in the caecum and a decreasing content in the colon. Time of adaptation of microbiota was also shown by xylanase and cellulase activities that were not detected until day 7 and 42, respectively. Results obtained shows that pig gut microbiota need a relative long time to adapt after a change of diet that could last up to 6 weeks. We were not able to detect a clear effect of dietary fibre content on this process.     

Counts of selected strict and facultatively anaerobic microorganisms in the crop and appendix of chickens and their adhesion characteristics]

The counts of strictly anaerobic amylolytic and lactate-utilizing bacteria, streptococci, lactobacilli, Escherichia coli and lactoso-negative bacteria were followed in chicks at the age of three to seven weeks. Age dependences of the counts in the given groups were investigated. An increase in the counts of amylolytic bacteria was observed in the caecum contents in the seventh week (4.63 +/- 0.322), and on the other hand a decrease in their counts in the caecum wall in the seventh week (3.30 +/- 0.833). The counts of E. coli adherent to the caecum wall also showed a decreasing tendency. Lactobacilli and streptococci were a stable component of the microflora of craw wall, in contrast with the decreasing counts of anaerobic amylolytic and lactate-utilizing bacteria. The average adherence index of lactobacillum isolates for adherence to the epithelial cells of craw or caecum wall ranged from 7.433 +/- 1.521-11.866 +/- 2.661, and/or 4.366 +/- 1.373-9.70 +/- 0.935. The average adherence index of the Propionibacterium acnes ranged from 7.766 +/- 0.408 to 17.40 +/- 4.721, and/or 5.10 +/- 0.738 to 7.60 +/- 1.784.     

Influence of a probiotic Enterococcus faecium strain on selected bacterial groups in the small intestine of growing turkey poults.

A feeding trial was carried out with turkey poults, which were fed a diet containing 10(10) viable probiotic E. faecium NCIB 10415 cells/kg feed. Samples of the intestinal tract were analyzed for lactate, colony forming units of total anaerobic bacteria, lactic acid bacteria, enterobacteria and enterococci. Furthermore, metabolic activity of total eubacterial, lactobacilli and enterococci was recorded in selected RNA-extracts with specific ribosomal RNA oligonucleotide probes. Animals fed the probiotic diet showed continously increasing lactate concentrations throughout the sampling period up to day 42 of life. No correlation was found for colony forming units (cfu) of lactic acid bacteria, but metabolic activity of lactobacilli showed very close relation to continously increasing lactate concentrations. Throughout the feeding trial, enterococci in the control group continously increased to a maximum of 10(4) cfu/g wet weight, but 10-fold higher enterococci cfu were generally found in the treated group. However, rRNA content as measure for metabolic activity showed a drastic decline in both groups after high metabolic activities on day 7. This study shows that E. faecium NCIB 10415 (E. faecium SF68) stimulates other lactic acid bacteria in the small intestine, especially lactobacilli.     

Exploring the use of molecular epidemiology to track bovine tuberculosis in Nigeria: an overview from 2002 to 2004

This study focused on the development of a reliable and cost-efficient DNA isolation procedure for the detection of Mycobacterium avium subsp. paratuberculosis (MAP) in faeces by previously developed IS900 and F57 quantitative real time PCR (qPCR) and their comparison with culture. The recovery of MAP DNA from the spiking experiments ranged from 29.1 to 102.4% of the input amount of MAP with median 37.9%. The limit of detection was determined to be 1.03 × 10(4) for F57 qPCR and 6.87 × 10(2)MAP cells per gram of faeces for IS900 qPCR, respectively. The developed technique for DNA isolation was coupled with IS900 qPCR and compared to traditional MAP culture using a cohort of 1906 faecal samples examined from 12 dairy cattle farms in our laboratory. From those 1906 original faecal samples, 875 were positive by IS900 qPCR and 169 by culture. None of the culture positive samples was negative by IS900 qPCR. This data facilitated development of a predictive model capable of estimating the probability of being culture positive by estimating the absolute number of MAP per gram of faeces as determined IS900 qPCR without performing the culture.     

伪狂犬病病毒野毒荧光定量PCR检测方法的建立

根据伪狂犬病病毒gE基因的序列,设计和合成了一对特异的可用于检测伪狂犬病病毒野毒的PCR引物和一条Taqman荧光探针,采用Li ght Cycl e 480荧光定量PCR仪,建立了一种可实时定量检测伪狂犬病病毒野毒的荧光定量PCR技术。该方法的线性范围为1.0×102～1.0×1010拷贝,灵敏度可达4拷贝。检测速度快,仪器的运行时间仅为1 h。对13株猪伪狂犬病病毒野毒进行了检测,结果均为阳性;与伪狂犬病gE基因缺失疫苗、猪细小病毒和鸭瘟病毒无非特异性反应。与病毒分离培养比较,该方法具有快速、灵敏、特异、定量、重复性好等优点,可望用于临床上伪狂犬病病毒野毒与疫苗毒的区分,伪狂犬病病毒野毒的检测和病毒分布的研究等。     

Results of cytologic and microbiologic analysis of bronchoalveolar lavage fluid in New Zealand White rabbits

OBJECTIVE: To determine cytologic and microbiologic findings in bronchoalveolar lavage (BAL) fluid and SpO(2) values obtained during BAL in healthy rabbits. ANIMALS: 9 rabbits. PROCEDURES: Bronchoscopic BAL of left and right caudal lobar bronchi (LB2 and RB4) was performed with 3 mL of sterile saline (0.9% NaCl) solution; SpO(2) was measured before, during, and after BAL. Percentage fluid recovered, total leukocyte counts, and differential cell counts were determined. Aerobic and anaerobic bacterial, mycoplasmal, and fungal cultures were performed from combined LB2 and RB4 samples. RESULTS: Mean +/- SD percentage fluid volumes recovered from LB2 and RB4 were 53 +/- 13% and 63 +/- 13%, respectively. Mean +/- SD total leukocyte counts from LB2 and RB4 were 422 +/- 199 cells/microL and 378 +/- 97 cells/microL, respectively. Macrophages were most frequently identified. There were no significant differences in volumes retrieved, total leukocyte counts, or differential cell percentages between LB2 and RB4. Microbial culture results were negative for 3 rabbits and positive for mixed aerobic and anaerobic bacterial growth in 6 and 2 rabbits, respectively. The SpO(2) was > or = 95% in 7 of 9 rabbits after anesthetic induction, < 95% in 5 of 6 rabbits 1 minute after BAL, and > or = 95% in 5 of 9 rabbits and > 90% in 4 of 9 rabbits 3 minutes after BAL. CONCLUSIONS AND CLINICAL RELEVANCE: Bronchoscopic BAL with 3 mL of saline solution provided adequate fluid recovery for microbiologic and cytologic examination from the caudal lung lobes. Transient low SpO(2) was detected immediately after BAL.     

Vaginal and uterine microflora of adult dogs

Aerobic and anaerobic microflora were identified and quantitated in 82 vaginal and 78 uterine samples obtained from mature bitches during different stages of the estrous cycle. The mean +/- SD of total bacterial counts/100 mg of vaginal contents of the 82 bitches was log 5.0 +/- 1.5, ranging from log 2.4 to log 8.8. The count at the estrous stage (log 7.8 +/- 0.7) was significantly higher (P less than 0.05) than that at the anestrus (log 4.4 +/- 1.0), pregnancy (log 5.9 +/- 1.3), and postpartum (log 5.1 +/- 1.5) stages. The common organisms isolated from the vaginas were Bacteroidaceae, streptococci, Pasteurella spp, and mycoplasmas. Organisms were isolated from 48 (68%) of 78 uterine samples. The range of total counts/100 mg of uterine contents was from log 1.6 to log 8.3. Staphylococci and mycoplasmas were frequently isolated from the uterine contents. Although many uterine microfloras were similar to vaginal microfloras, some uterine culture had a single isolate identified. There were no pathologic findings in most of the uteri. Seemingly, vaginal bacteria frequently flow into the uterus, yet they rarely cause uterine infection.     

Effect of sample pooling and transport conditions on the clinical sensitivity of a real-time polymerase chain reaction assay for Campylobacter fetus subsp. venerealis in preputial samples from bulls

The diagnosis of bovine genital campylobacteriosis (BGC) presents significant challenges, as traditional methods lack sensitivity when prolonged transport of samples is required. Assays of preputial samples by means of real-time polymerase chain reaction (PCR) provide good sensitivity and high throughput capabilities. However, there is limited information on the acceptable duration of transport and temperature during transport of samples. In addition, the use of pooled samples has proven to be a valuable strategy for the diagnosis of other venereal diseases in cattle. The objectives of the present study were to determine the effect of sample pooling and of transport time and temperature on the clinical sensitivity of a real-time quantitative PCR (qPCR) assay for Campylobacter fetus subsp. venerealis in preputial samples from beef bulls. Eight infected bulls and 176 virgin yearling bulls were used as the source of samples. The qPCR sensitivity was comparable for unpooled samples and pools of 5 samples, whereas sensitivity was decreased for pools of 10 samples. Sensitivity for the various pool sizes improved with repeated sampling. For shorter-term transport (2 and 48 h), sensitivity was greatest when the samples were stored at 4°C and 30°C, whereas for longer-term transport (96 h) sensitivity was greatest when the samples were stored at −20°C. The creation of pools of 5 samples is therefore a good option to decrease costs when screening bulls for BGC with the qPCR assay of direct preputial samples. Ideally the samples should be stored at 4°C and arrive at the laboratory within 48 h of collection, but when that is not possible freezing at −20°C could minimize the loss of sensitivity.     

Evaluation the effect of subchronic feeding of transgenic cotton line (CKC1) on the faecal microbiota of albino rabbits

Recent studies have demonstrated a strong relationship between the intestinal microbiota and the host health. As such, consumers are increasingly becoming more concerned about the potential effect of certain foods/feeds, particularly of transgenic origin on the gut microbiota. Although the European Food Safety Authority has recommended in their guidelines, to study the effect of transgenic food/feed on host-microbiota, yet, few studies have focused on the evaluation of such effects mainly due to culturing difficulties. Therefore, this study was intended to evaluate the potential adverse effects of transgenic diet consumption on some specific gut microflora (Lactobacillus group, Bifidobacterium genus, Escherichia coli subgroup and Enterococcus genus) of rabbits. A total of forty-eight rabbits were randomly assigned into four groups and fed a diet containing a variable proportion of transgenic cottonseeds at 0, 20, 30 and 40% inclusion level, respectively. Changes in the specific or total faecal bacterial population were monitored at five different experimental stages (i.e. 0, 45, 90, 135 and 180 days) using both the traditional plate count method (TM) and quantitative real-time PCR (qPCR). No significant differences (p > .05) were observed concerning numbers of specific bacteria or total bacteria between the control and experimental groups, though qPCR showed numerically higher values in terms of 16S rRNA gene copies as compared to the values obtained from TM. However, such numerical differences were biologically insignificant (p > .05). Similarly, no significant variations were noticed in the calculated B/E (log₁₀ copies of Bifidobacterium per g faces/log10 copies of E. coli genome per g faeces) ratios in all the groups. All the ratios were in the range of 1.24 to 1.30 throughout the experiment, indicating a good balance of intestinal microflora and greater resistance to intestinal disorders. It is therefore concluded that feeding transgenic cottonseeds could not adversely affect the gut microflora of rabbits during a long-term study.     

Development and Comparison of Sampling Techniques for a Broad Range,Semiquantitative Polymerase Chain Reaction Assay for Detection of Bacterial DNA in the Equine Uterus

Microbial culture from a double-guarded culture swab is commonly used to diagnose infectious endometritis. The objective of this study was to develop a quantitative polymerase chain reaction (qPCR) assay to detect a broad range of bacteria from equine uterine samples. Twenty-seven mares with a clinical history of endometritis had a double-guarded culture swab collected for analysis by qPCR and microbial cultures. An additional 12 mares had a uterine biopsy sample collected for qPCR analysis, microbial culture, and histopathology. Subsequently, a double-guarded culture swab for microbial culture and a cytology brush sample were also collected. The qPCR assay detected bacterial DNA in nine of 27 mares from a double-guarded swab and six of 12 mares from an endometrial biopsy. Positive microbial growth was detected in nine of 27 mares and four of 12 mares from a double-guarded culture swab. Bacterial DNA was detected in two of 27 mares and two of 12 mares without subsequent microbial growth. The simple presence of an organism's DNA allows for detection by nonculture-based systems, both live and dead organisms can be identified. In conclusion, the qPCR assay was determined to be a sensitive diagnostic technique for identifying pathogens associated with infectious endometritis. The primary application of the qPCR assay is detection of potential pathogenic bacteria in the uterus of a mare suspected of having infectious endometritis when a traditional microbial culture is negative. Further work is warranted to determine if mares positive for bacterial DNA and negative for microbial culture are affected clinically.     

Detection and quantification of Renibacterium salmoninarum DNA in salmonid tissues by real-time quantitative polymerase chain reaction analysis.

Renibacterium salmoninarum is an important salmonid pathogen that is difficult to culture. We developed and assessed a real-time, quantitative, polymerase chain reaction (qPCR) assay for the detection and enumeration of R. salmoninarum. The qPCR is based on TaqMan technology and amplifies a 69-base pair (bp) region of the gene encoding the major soluble antigen (MSA) of R. salmoninarum. The qPCR assay consistently detected as few as 5 R. salmoninarum cells per reaction in kidney tissue. The specificity of the qPCR was confirmed by testing the DNA extracts from a panel of microorganisms that were either common fish pathogens or reported to cause false-positive reactions in the enzyme-linked immunosorbent assay (ELISA). Kidney samples from 38 juvenile Chinook salmon (Oncorhynchus tshawytscha) in a naturally infected population were examined by real-time qPCR, a nested PCR, and ELISA, and prevalences of R. salmoninarum detected were 71, 66, and 71%, respectively. The qPCR should be a valuable tool for evaluating the R. salmoninarum infection status of salmonids.     

A cytolethal distending toxin gene-based multiplex PCR assay for detection of Campylobacter spp. in stool specimens and comparison with culture method

In this study, we evaluated the applicability of cytolethal distending toxin (cdt) gene-based species-specific multiplex PCR for the direct detection and identification of Campylobacter jejuni, C. coli and C. fetus from stool specimens of patients with gastroenteritis in comparison to culture methods. A total of 711 stool specimens were examined for the isolation or detection of campylobacters by using Skirrow's selective agar culture plates, a filtration method and the multiplex PCR assay. Forty-one and 36 C. jejuni strains were isolated by culture and filtration methods, respectively. In addition, 2 and 3 C. coli strains were isolated by Skirrow and the filtration methods, respectively. However, when the multiplex PCR was employed, the cdtB genes of C. jejuni and C. coli were detected in 45 and 4 stool samples, respectively, and 9 C. jejuni PCR-positive samples by multiplex PCR were negative by culture method. Sequence analysis of the PCR products obtained from 8 stool specimens from which campylobacters were not isolated by culture method but the sequences exactly matched with that of the cdtB gene of C. jejuni strain 81-176. None of the remaining stool samples which were culture negative for campylobacters produced any amplicon. Stool samples were defined as Campylobacter-positive if detected by any method. The sensitivity of the multiplex PCR was 83%, which was higher than Skirrow (74%) and filtration method (66%). These data indicate that cdtB gene-based multiplex PCR is a rapid and more sensitive method to identify the most important species of Campylobacter for human diseases. (248).     

Molecular ecological analysis of porcine ileal microbiota responses to antimicrobial growth promoters

Cultivation-independent microbial molecular ecology approaches were used to examine the effects of antibiotic growth promoters on the pig ileal microbiota. Five-week-old barrows were fitted with a simple T-cannula at the distal ileum. Three diets meeting or exceeding the minimum nutrient requirements were fed for 5 wk and supplemented as follows: 1) negative control (no antibiotic; n = 5), 2) continuous tylosin administration (n = 5), and 3) an antibiotic rotation sequence (wk 1, chlorotetracycline sulfathiazole penicillin; wk 2, bacitracin and roxarsone; wk 3, lincomycin; wk 4, carbadox; wk 5, virginiamycin; n = 5). Ileal luminal contents were collected for DNA isolation at the end of each of the 5 wk of the testing period. The V3 region of 16S rDNA was amplified by PCR and analyzed via denaturing gradient gel electrophoresis (DGGE) and quantitative polymerase chain reaction (qPCR). Resulting PCR-DGGE band numbers (bacterial species) were counted, and the banding patterns analyzed by calculating Sorenson's pairwise similarity coefficients (C(S)), an index measuring bacterial species in common among samples. Band numbers and total bacterial DNA concentrations decreased (P < 0.05) temporally in antibiotic-treated pigs compared with controls. Comparisons between treatments yielded low intertreatment C(S) indices, indicating treatment-dependent alterations in banding patterns, whereas intratreatment comparisons revealed increased homogeneity in antibiotic-treated vs. control pigs. Sequence analysis of treatment-specific bands identified three Lactobacillus, one Streptococcus, and one Bacillus species that were diminished with antibiotic rotation treatment, whereas tylosin selected for the presence of L. gasseri. Lactobacillus-specific qPCR was performed and analyzed as a percentage of total bacteria to further evaluate the effects of antibiotic administration on this genus. Total bacteria were decreased (P < 0.05) by tylosin and rotation treatments, whereas the percentage of lactobacilli increased (P < 0.05) by d 14 and through d 28 in tylosin-treated pigs. The decrease in total bacteria by antibiotics may reduce host-related intestinal or immune responses, which would divert energy that could otherwise be used for growth. Conversely, the ability of tylosin to improve animal growth may relate to its apparent selection for lactobacilli, commensals known to competitively exclude potentially pathogenic species from colonizing the intestine.     

布氏杆菌微滴数字PCR方法的建立

为建立一种布氏杆菌微滴数字PCR qPCR方法,对布氏杆菌病的定量诊断提供技术支持,在实时荧光PCR (qPCR)检测方法(T/CVMA 20-2020)的基础上,建立了布氏杆菌微滴数字PCR方法,并对方法的反应条件进行了优化,同时对其敏感性、特异性、重复性进行了评估.结果 显示:本方法的最低检测下限为2.6 copi...     

The prevalence and PCR detection of Salmonella contamination in raw poultry

Contaminated poultry meat has been identified as one of the principal foodborne sources of Salmonella. The development of rapid detection assays for Salmonella would enable official agencies and food industries to identify contaminated foodstuffs in a more timely manner. In addition, these diagnostic tools could allow more 'real time' decisions to be made regarding end product acceptability. In this study, a survey was carried out to determine the prevalence of Salmonella in raw broiler carcasses. A total of 198 neck skin samples were obtained from within 40 flocks at a commercial broiler slaughtering facility. The presence of Salmonella was assessed by traditional culture methods and by a Salmonella-specific polymerase chain reaction (PCR) test. Salmonella was recovered from 32 (16%) of all samples using traditional culture methods. In contrast, the PCR assay proved to be more sensitive and detected Salmonella DNA in 38 (19%) of the samples tested. The pathogen was detected in 45 (23%) of the 198 samples when culture and PCR results were combined. The sensitivity of the PCR test was also greater than culture when detecting Salmonella from within flocks (53% of flocks by PCR, 30% of flocks by culture). The combination of both tests revealed that 55% of the flocks were contaminated with Salmonella. The PCR assay proved to be a highly specific and sensitive method for detecting Salmonella and the incorporation of a routine PCR test in conjunction with standard culture could be effective in providing a more accurate profile of the prevalence of this pathogen in broiler carcasses.