Evaluation of PCR inhibitory effect of enrichment broths and comparison of DNA extraction methods for detection of Salmonella Enteritidis using real-time PCR assay

The best enrichment broth and DNA extraction scheme was determined for rapid and sensitive detection of Salmonella Enteritidis in steamed pork using real-time PCR. The inhibitory effect of commonly used Salmonella enrichment broths, Rappaport-Vassiliadis (RV) and Muller-Kauffmann tetrathionate with novobiocin (MKTTn), on real-time PCR was confirmed. The inhibition of PCR was statistically significant (p < 0.05) in RV and MKTTn, as compared with buffered peptone water (BPW) or phosphate-buffered saline. The inhibitory effect of the selective enrichment media was successfully removed by using a modified DNA extraction, PrepMan Ultra Reagent with an additional washing step or the DNeasy Tissue Kit. In three experiments, when applied to detection of Salmonella Enteritidis in steamed pork, the real-time PCR coupled with single 24 h enrichment with BPW performed better than double 48 h enrichment with BPW plus RV or MKTTn. The simple real-time PCR assay using BPW proved to be a rapid and sensitive test for detection of low concentrations of Salmonella Enteritidis in steamed pork samples as compared with the conventional culture method.     

Comparison of bacterial enriched-broth culture, enzyme linked immunosorbent assay, and broth culture-polymerase chain reaction techniques for identifying asymptomatic infections with Salmonella in swine

A polymerase chain reaction (PCR) assay was combined with a broth-culture enrichment system to detect Salmonella shed in feces from subclinically infected swine. The effectiveness of the broth culture-polymerase chain reaction (BC-PCR) assay to identify pigs shedding Salmonella in feces was compared with a microbiological culture and a commercial enzyme linked immunosorbent assay (ELISA) kit to detect Salmonella-specific serum antibody. A total of 67 pigs were tested by each of the 3 methodologies. Forty-one pigs tested positive for Salmonella by BC-PCR and ELISA identified 6 positives and 23 suspicious samples. It was shown that the BC-PCR assay is a rapid diagnostic tool for detecting of Salmonella shed by asymptomatic swine compared with current diagnostic technologies.     

The effect of environmental poultry samples on the pH of typical Salmonella pre-enrichment and enrichment media following incubation

The first step to detect Salmonella in feed and other dry contaminated samples is a pre-enrichment broth that can assist in the recovery of small numbers of stressed Salmonella cells. A previous study demonstrated that incubation of feed and feed ingredients in commonly used pre-enrichment media resulted in a low pH that injured or killed the Salmonella. The objective of this study was to determine which environmental samples and pre-enrichment and selective broths could interfere with the accuracy of Salmonella detection by allowing pH to drop. Samples were collected from commercial poultry operations. Triplicate 10-g subunits were dispensed into sterile 18-oz Whirl Pak bags and 90 mL of each of the media [lactose broth (LB), buffered peptone water (BPW), Universal Pre-enrichment (UP), minimal salts (M-9), tetrathionate broth (TT) and Rappaport-Vassiliadis broth (RV)] were added to the bags and incubated 24 h at 37°C (or 42°C for RV and TT). The pH was measured after incubation. With turkey litter, fluff, and eggshells, regardless of media, the pH never went below 5.7. Regardless of sample type, the lowest pH was 6.1, 6.2, and 6.4 for UP, M-9, and BPW, respectively. For the pre-enrichment medium commonly used in the US, (LB) the pH dropped to 4.7 to 4.9 with broiler litter and 4.2 for boot covers used in turkey or broiler houses. Many researchers testing these sample types are unaware of this potential for pH change during pre-enrichment and may be underestimating the presence of Salmonella.     

Effects of sample storage and delayed secondary enrichment on detection of Salmonella spp in swine feces

OBJECTIVE: To determine effects of fecal sample storage and delayed secondary enrichment (DSE) on detection of Salmonella spp in swine feces. Sample Population-Fecal samples obtained from 84 pigs in a commercial herd. PROCEDURE: Each fecal sample underwent 3 storage treatments: no storage (ie, processed on the day of collection), storage at 4 C for 6 days, and storage at -15 C for 14 days. After assigned storage treatments, all samples were enriched in Rappaport-Vassiladias (RV) broth (single enrichment) and plated on XLT4 agar. Delayed secondary enrichment was performed, using single enrichment broths that were stored for 4 days at room temperature. RESULTS: Of 504 cultures, 186 (36.9%) were Salmonella positive. A difference in proportions of samples with positive results was not found between same-day processing and storage at 4 C for 6 days. Compared with use of single enrichment for 24 hours (34% positive), use of DSE resulted in a greater proportion (40%; P < 0.001) of samples with positive results. Estimated relative sensitivities for the storage methods were 0.90, 0.85, and 0.71 for same-day processing, storage at 4 C for 6 days, and storage at -15 C for 14 days, respectively. CONCLUSIONS: Where practical, processing of fecal samples on the day of collection is recommended, although storage at 4 C for several days does not result in marked loss of sensitivity. Improved detection associated with DSE warrants further investigation and optimization.     

Epidemiological associations between characteristics of registered broiler chicken flocks in Canada and the Salmonella culture status of floor litter and drinking water

A Canada-wide flock management survey of 294 randomly selected commercial broiler chicken flocks was conducted during 1989-1990. The prevalence of flocks that yielded Salmonella from cultures of floor litter or drinking water, and the prevalence of floor litter samples that yielded Salmonella, were significantly associated (p < 0.05) with the age of the flock and the region of Canda in which the flock was located. Culture of Salmonella from the drinking water was significantly associated (p < 0.05) with the type of drinker used, the dead bird disposal method, and the region of Canada in which the flock was located. There was a significantly greater risk (p < 0.05) of contamination of drinking water with Salmonella from trough drinkers (odds ratio = 7.99) and plastic bell drinkers (odds ratio = 6.10) than from nipple drinkers. No significant associations were found between pest control, restrictions on visitors, clean-out methods, or water sanitization and the culture of salmonellae from floor litter or drinking water.     

Relationship between the Lactic Acid Content and Sour Taste of Broiler Broth and the Broth of Choshu-Kurokashiwa—a Japanese Jidori Chicken

The present study aimed to determine whether lactic acid content is associated with the intensity of the sour taste of Choshu-Kurokashiwa broth. Chicken broth was prepared using breast (pectoralis major) and leg (thigh + drumstick) meat of male and female Choshu-Kurokashiwa and broilers. These broths were assessed by a screened and trained panel and analyzed for sour taste substances (lactic, citric, pyruvic, malic, succinic, acetic, phosphoric, aspartic, and glutamic acids) and pH. The sensory sour taste was significantly higher in the Choshu-Kurokashiwa breast broth than in the broiler breast broth (P>0.001), and no significant difference was observed in the leg broths among the breeds (P<0.05). Choshu-Kurokashiwa breast broth had a significantly higher lactic acid content than broiler breast broth (P<0.001). The leg meat broth of male Choshu-Kurokashiwa had a significantly lower lactic acid content than that of female Choshu-Kurokashiwa and broiler leg meat (P<0.01). The sensory sour taste score was significantly and positively correlated with lactic acid content in the breast broth (P<0.001), but not in the leg broth (P<0.1). No other organic acids were detected. Phosphoric acid and glutamic acid contents were higher in broiler broth than in Choshu-Kurokashiwa broth for both breast and leg meat (P<0.001). In the breast broth, the aspartic acid content was not significantly different (P>0.1), and in the leg broth, it was higher in broiler and female Choshu-Kurokashiwa broth than in male Choshu-Kurokashiwa broth (P<0.001). The present study suggests that high lactic acid content contributes to the sour taste of the Choshu-Kurokashiwa breast broth and demonstrated that the lactic acid content is an essential indicator for determining the sour taste of Choshu-Kurokashiwa breast meat.     

Comparison of wet-mount,Wright-Giemsa and Gram-stained urine sediment for predicting bacteriuria in dogs and cats

This study assessed the standard urinalysis technique and sediment stain techniques as predictors of bacterial culture results for canine and feline urine. Canine (n = 111) and feline (n = 79) urine samples were evaluated using unstained wet-mount and air-dried Gram and Wright-Giemsa stained sediment; results were compared to aerobic bacterial culture. Eleven canine and 7 feline urine samples were culture positive. Unstained wet-mount and stained sediment had sensitivities of 89% and 83% and specificities of 91% and 99%, respectively. The specificity of using either stain was higher (P < 0.01) than wet-mount examination for detecting bacteriuria. There were significant differences among 3 technologists in detecting true positives (P < 0.01). Association of sediment and culture results used 112 canine and 81 feline samples. There was a negative association (P < 0.01) between lipid detection and wet-mount identification of bacteria.     

Comparison of milk culture, direct and nested polymerase chain reaction (PCR) with fecal culture based on samples from dairy herds infected with Mycobacterium avium subsp. paratuberculosis

Mycobacterium avium subsp. paratuberculosis (MAP) is the etiologic agent of Johne’s disease in cattle and other farm ruminants, and is also a suspected pathogen of Crohn’s disease in humans. Development of diagnostic methods for MAP infection has been a challenge over the last few decades. The objective of this study was to investigate the relationship between different methods for detection of MAP in milk and fecal samples. A total of 134 milk samples and 110 feces samples were collected from 146 individual cows in 14 MAP-infected herds in southwestern Ontario. Culture, IS900 polymerase chain reaction (PCR) and nested PCR methods were used for detecting MAP in milk; results were compared with those of fecal culture. A significant relationship was found between milk culture, direct PCR, and nested PCR (P < 0.05). The fecal culture results were not related to any of the 3 assay methods used for the milk samples (P > 0.10). Although fecal culture showed a higher sensitivity than the milk culture method, the difference was not significant (P = 0.2473). The number of MAP colony-forming units (CFU) isolated by culture from fecal samples was, on average, higher than that isolated from milk samples (P = 0.0083). There was no significant correlation between the number of CFU cultured from milk and from feces (Pearson correlation coefficient = 0.1957, N = 63, P = 0.1243). The animals with high numbers of CFU in milk culture may not be detected by fecal culture at all, and vise versa. A significant proportion (29% to 41%) of the positive animals would be missed if only 1 culture method, instead of both milk and feces, were to be used for diagnosis. This suggests that the shedding of MAP in feces and milk is not synchronized. Most of the infected cows were low-level shedders. The proportion of low-level shedders may even be underestimated because MAP is killed during decontamination, thus reducing the chance of detection. Therefore, to identify suspected Johne’s-infected animals using the tests in this study, both milk and feces samples should be collected in duplicate to enhance the diagnostic rate. The high MAP kill rate identified in the culture methods during decontamination may be compensated for by using the nested PCR method, which had a higher sensitivity than the IS900 PCR method used.     

Functional amino acid supplementation,regardless of dietary protein content,improves growth performance and immune status of weaned pigs challenged with Salmonella Typhimurium

High dietary protein may increase susceptibility of weaned pigs to enteric pathogens. Dietary supplementation with functional amino acids (FAA) may improve growth performance of pigs during disease challenge. The objective of this study was to evaluate the interactive effects of dietary protein content and FAA supplementation above requirements for growth on performance and immune response of weaned pigs challenged with Salmonella. Sixty-four mixed-sex weanling pigs (13.9 ± 0.82 kg) were randomly assigned to dietary treatments in a 2 × 2 factorial arrangement with low (LP) or high protein (HP) content and basal (AA–) or FAA profile (AA+; Thr, Met, and Trp at 120% of requirements) as factors. After a 7-d adaptation period, pigs were inoculated with either a sterile saline solution (CT) or saline solution containing Salmonella Typhimurium (ST; 3.3 × 10⁹ CFU/mL). Growth performance, body temperature, fecal score, acute-phase proteins, oxidant/antioxidant balance, ST shedding score in feces and intestinal colonization, fecal and digesta myeloperoxidase (MPO), and plasma urea nitrogen (PUN) were measured pre- and postinoculation. There were no dietary effects on any measures pre-inoculation or post-CT inoculation (P > 0.05). Inoculation with ST increased body temperature and fecal score (P < 0.05), serum haptoglobin, plasma superoxide dismutase (SOD), malondialdehyde (MDA), PUN, and fecal MPO, and decreased serum albumin and plasma reduced glutathione (GSH):oxidized glutathione (GSSG) compared with CT pigs (P < 0.05). ST-inoculation reduced average daily gain (ADG) and feed intake (ADFI) vs. CT pigs (P < 0.05) but was increased by AA+ vs. AA– in ST pigs (P < 0.05). Serum albumin and GSH:GSSG were increased while haptoglobin and SOD were decreased in ST-inoculated pigs fed AA+ vs. AA– (P < 0.05). PUN was higher in HP vs. LP-fed pigs postinoculation (P < 0.05). Fecal ST score was increased in ST-inoculated pigs on days 1 and 2 postinoculation and declined by day 6 (P < 0.05) in all pigs while the overall score was reduced in AA+ vs. AA– pigs (P < 0.05). Cecal digesta ST score was higher in HP vs. LP-fed pigs and were lower in AA+ compared with AA– fed pigs in the colon (P < 0.05). Fecal and digesta MPO were reduced in ST pigs fed AA+ vs. AA– (P < 0.05). These results demonstrate a positive effect of FAA supplementation, with minimal effects of dietary protein, on performance and immune status in weaned pigs challenged with Salmonella.     

Evaluation of oral fluids for surveillance of foodborne and zoonotic pathogens in pig farms

The use of oral fluid (OF) to detect zoonotic pathogens in pigs has been only scarcely assessed. We evaluated OF as a potential specimen for detection by culture of methicillin-resistant Staphylococcus aureus (MRSA) and Yersinia enterocolitica, and the detection of antibodies against Salmonella spp. and hepatitis E virus (HEV) using commercial ELISAs. Samples from 33 pig farms were collected at the beginning and end of the fattening period. Results of the OF samples were compared with the results of serum samples and nasal swabs from individual pigs and pen floor fecal samples, using the Cohen kappa (κ) and the McNemar test. For Salmonella spp. antibodies, OF samples were negative, although the corresponding serum samples were positive. The detection of HEV antibodies in sera and OF had agreement at the first sampling, and poor and significant agreement at the second sampling (κ = 0.185, McNemar p = 0.238; κ = 0.088, McNemar p < 0.001). At both sampling times, the detection of MRSA in nasal swabs and OF showed agreement (κ = 0.466, McNemar p = 0.077; κ = 0.603, McNemar p = 1); agreement was seen for the detection of Y. enterocolitica in fecal and OF samples (κ = 0.012, McNemar p = 0.868; κ = 0.082, McNemar p = 0.061, respectively). According to the McNemar test, the use of pen-based OFs is more feasible for the detection of MRSA and Y. enterocolitica by culture than is detection of antibodies by commercial ELISA.     

Salmonella isolated from individual reptiles and environmental samples from terraria in private households in Sweden

Background

This study investigates Salmonella spp. isolated from privately kept reptiles and from environmental samples such as bedding materials or water from the floor of the enclosures (terraria). It also compares isolation of Salmonella using Modified Semisolid Rappaport-Vassiliadis (MSRV) medium or selective enrichment in Rappaport-Vassiliadis-Soya (RVS) pepton broth. Cloacal swabs or swabs from the cloacal area were collected from 63 individual reptiles belonging to 14 households. All reptiles were from different terraria and from 62 of these, environmental samples were also collected. Sampling were done by the reptile owners according to written instructions and sent by mail immediately after sampling. All but three samples were analyzed within 24 h after collection. Colonies suspected for Salmonella were tested for agglutination and serotyped using the White-Kauffmann-Le Minor scheme. The relative sensitivity (se) and specificity (sp) for MSRV compared with RVS, and the agreement coefficient kappa (κ) were calculated.

Results

Salmonella was isolated from 50/63 (80%) terraria, either from the reptiles (31/63; 49%) or from bedding material (39/62; 63%). The most common subspecies was Salmonella enterica subspecies enterica followed by S. enterica subspecies diarizonae. In reptiles, the most common S. enterica subspecies enterica serovars were Java (n = 4) and Fluntern (n = 4), compared with the serovars Tennessee (n = 10) and Fluntern (n = 10) in the environmental samples. The exact same set of Salmonella subspecies and serovars were not isolated from the individual reptiles and the environmental samples from any of the households. Isolation using MSRV yielded more Salmonella isolates 61/113 (54%) than enrichment in RVS 57/125 (46%). The se was 97.9% (95% Confidence Interval 93.9-100), the sp 78.5% (95% CI 68.5-88.5) and the κ 0.74, indicating substantial agreement between the tests.

Conclusions

Salmonella can be expected to be present in environments where reptiles are kept. This constitutes public health risks and should be considered during handling of the reptiles and during cleaning and disposal of bedding. A combination of different culturing techniques may be used to increase the isolation rate.     

Evaluation of the specificity of three enzyme-linked immunosorbent assays for detection of antibodies against Salmonella in bovine bulk milk

Background

The Swedish Salmonella control program has been running for decades and has resulted in a low prevalence of Salmonella in Swedish food producing animals. Routine bacteriology is used to detect Salmonella, however, bacteriology is time consuming, costly and has a low sensitivity. Different enzyme-linked immunosorbent assays (ELISAs) have been developed for detection of antibodies against Salmonella Dublin and S. Typhimurium in bovine bulk milk, individual milk samples as well as in sera. Screening bulk milk for antibodies against Salmonella spp. could improve the cost-effectiveness of the surveillance in Swedish dairy cattle, but as characteristics of tests may vary in different populations, tests should always be evaluated in the specific population where they will be used. Hence, the aim of this study was to evaluate the specificities of three bovine ELISAs when used to analyse bulk milk samples from Swedish dairy cattle. A second aim was to compare the performance of the two Dublin ELISAs tested.

Methods

Bulk milk samples for analysis were randomly selected from samples collected within the Swedish bulk milk sampling scheme and analyzed with the three ELISAs; a Danish in-house Dublin ELISA, PrioCHECK® Salmonella Ab bovine Dublin ELISA and PrioCHECK® Salmonella Ab bovine ELISA (hereafter named mixed ELISA). The specificities of the ELISAs were calculated assuming a disease-free status in Sweden i.e. that all test positive samples were assumed to be false positive results. This assumption can be used when a disease is known to be infrequent.

Results

The calculated specificities of the two Dublin ELISAs and the mixed ELISA, when using the producer’s recommended cut-off value of the corrected optic-density percent (ODC%) were 99.4% (95% Confidence Interval (CI): 98.8% -99.8%), 99.4% (95% CI: 98.8% -99.8%) and 97.9% (95% CI: 96.8% -98.7%), respectively. The correlation between the ODC% values of the two Dublin ELISAs was 0.83.

Conclusions

We conclude that the evaluated ELISAs have sufficiently high specificities to be used as supplement to bacteriological examinations in the Swedish Salmonella control program in cattle as well as a primary screening test in routine surveillance for S. Dublin.     

A longer adaptation period to a functional amino acid-supplemented diet improves growth performance and immune status of Salmonella Typhimurium-challenged pigs

We recently showed that dietary supplementation with key functional amino acids (FAA) improves growth performance and immune status of Salmonella Typhimurium (ST)-challenged pigs. It is not known if ST-challenged pigs will benefit from a longer adaptation period to FAA. The objective of this study was to evaluate the effects of different adaptation periods to diets containing FAA above requirements for growth on performance and immune response of weaned pigs subsequently challenged with ST. A total of 32 mixed-sex weanling pigs (11.6 ± 0.3 kg) were randomly assigned to 1 of 4 dietary treatments, being a basal amino acid (AA) profile fed throughout the experimental period (FAA−) or a functional AA profile (FAA+; Thr, Met, and Trp at 120% of requirements) fed only in the postinoculation (FAA+0), for 1 wk pre- and postinoculation (FAA+1), or throughout the experimental period (FAA+2). After a 14-d adaptation period, pigs were inoculated with ST (2.15 × 10⁹ CFU/mL). Growth performance, body temperature, fecal score, acute-phase proteins, oxidant/antioxidant balance, score for ST shedding in feces and intestinal colonization, and fecal and digesta myeloperoxidase (MPO) were measured pre- and postinoculation. Postinoculation body temperature and fecal score, serum haptoglobin, plasma superoxide dismutase (SOD), malondialdehyde (MDA), and fecal MPO were increased while serum albumin and plasma reduced glutathione (GSH):oxidized glutathione (GSSG) were reduced compared to pre-inoculation (P < 0.05). Average daily gain and G:F were greater in FAA+2 pigs compared to FAA− pigs (P < 0.05). Serum albumin was higher in FAA+2 and FAA+1 compared to FAA+0 and FAA− pigs (P < 0.05) while FAA+2 pigs had lower haptoglobin compared to FAA− (P < 0.05). Plasma SOD was increased and GSH:GSSG was decreased in FAA− pigs compared to the other treatments (P < 0.05). Score for ST shedding in feces was progressively lower from d 1 to 6 regardless of treatment (P < 0.05) and was lower in FAA+2 pigs compared to FAA− and FAA+0 (P < 0.05). Counts of ST in colon digesta were higher in FAA− and FAA+0 pigs compared to FAA+2 (P < 0.05). Fecal and colonic digesta MPO were lower in FAA+2 and FAA+1 pigs compared to FAA− (P < 0.05). These results demonstrate a positive effect of a longer adaptation period to FAA-supplemented diets on performance and immune status of weaned pigs challenged with Salmonella.     

The Application of Copper Waterline on Laying Performance and Gut Health of Aged Laying Hens

The effect of the application of copper waterline on the performance and gut health of aged laying hens was evaluated in this study. Forty-eight 70-week-old laying hens were divided into two groups (three replicates of eight hens each): control and copper (Cu) groups provided with normal polyvinyl chloride (PVC) waterline or Cu waterline. The laying performance was measured during the four-week period of the experiment. The intestinal antioxidant status and the microbiota diversity of the cecal content were determined. Moreover, a bacteriostasis test on Escherichia coli and Salmonella enteritidis was conducted after inoculation in waterline and hens, respectively. The water Cu²⁺ content was increased by Cu waterline compared to the control (P<0.05). Cu waterline had no detectable effect on most production performances, however, it increased the egg weight (P<0.05). Cu waterline increased the Cu level in the eggshell. Cu level in excreta increased with time, especially in the final two weeks, however, there was no significant change in fecal Cu excretion. The lipid peroxidation product malondialdehyde content in ileum decreased (P<0.01), while the activities of CuZn-superoxide dismutase (SOD) of ileum and glutathione peroxidase (GSH-PX) activity of jejunum and ileum increased after Cu treatment. The relative abundance and richness of cecal microbiota increased after Cu treatment (P<0.05). Cu waterline changed the microbial composition, including the increased proportion of Methanocorpusculum, Paludibacter, and decreased proportion of Fucobacterium, Anaerobiospirillum, and Campylobacter. The colonization of E. coli and S. enteritidis in Cu waterline was suppressed by Cu treatment, indicating that Cu waterline had potential antibacterial properties. The result suggests that Cu waterline could inhibit the colonization of pathogenic microorganisms such as E. coli and Salmonella and facilitate the enrichment of cecal microbiota diversity.     

Salmonella Isolated from Animals and Feedstuffs in Sweden during 1988-1992

The present paper surveys the number of Salmonella isolations in animals and feedstuffs in Sweden during 1988–1992. It is the eighth in a series of reports published by the National Veterinary Institute (NVI) since 1949. During the period referred to, 602 outbreaks of Salmonella were reported in animals, both domestic and wild. Compared with the previous 5-year period there was a 20% reduction in the number of outbreaks (760). Fifty-six different serotypes were reported, 19 of which had never been isolated in any animal in Sweden previously. A temporary increase in the number of outbreaks in poultry was seen in 1991 following an extended sampling before slaughter of layers. A remarkably high prevalence (38%) of Salmonella was observed in snakes in the wild.In 1990, the end-point testing of feeds was replaced by an approach based on HACCP (Hazard Analysis Critical Control Point) principles for the monitoring of feed mills. Significantly higher number of Salmonella positive samples were found by using this technique compared with the previous analysis of finished feed.It is concluded that the adopted Salmonella control program has contributed to a reduced number of Salmonella outbreaks in animals in Sweden.     

Simplified isolation and enrichment of spermatogonial stem-like cells from pubertal domestic cats (Felis catus)

The efficiency of spermatogonial stem cell (SSC) isolation and culture from pubertal donors is currently poor primarily, because of contamination with other testicular cells. This study aimed to purify SSC-like cells using different extracellular matrixes and a discontinuous gradient density. In experiment 1, testes (n=6) were analyzed for histology and SSC-related protein expressions (laminin, SSEA-4, DDX-4 and GFRα-1). After enzymatic digestion, the cell suspension was plated onto either a laminin- or gelatin-coated dish. The number of SSC-like cells was determined at 15, 30 and 60 min of culture (experiment 2). Experiment 3 was performed to test whether or not the additional step of Percoll gradient density centrifugation could really improve purification of SSC-like cells. Testicular histology revealed complete spermatogenesis with laminin expression essentially at the basal lamina of the seminiferous tubules. SSEA-4 and GFRα-1 co-localized with DDX-4 in the spermatogonia. The relative percentage of SSC-like cells, as determined by cells expressing SSEA-4 (59.42 ± 2.18%) and GFRα-1 (42.70 ± 1.28%), revealed that the highest SSC-like cell purity was obtained with the 15-min laminin-coated dish compared with other incubation times and gelatin treatment (P<0.05). Percoll treatment prior to laminin selection (15 min) significantly improved SSC-like cell recovery (91.33 ± 0.14%, P<0.001) and purity (83.82 ± 2.05% for SSEA-4 and 64.39 ± 1.51% for GFRα-1, P<0.05). These attached cells demonstrated a typical SSC-like cell morphology and also expressed POU5F1, RET and ZBTB16 mRNA. In conclusion, double enrichment with Percoll gradient density centrifugation and laminin plating highly enriched the SSC-like cells population.     

Detection and characterization ofSalmonella typhimurium from a dairy herd in North Dakota

Nasal secretions, faecal samples and buffy coats were obtained from 102 cattle from a North Dakota dairy herd with a history of calf scours. Treated buffy coats, faecal samples and nasal secretions were inoculated into tetrathionate broth (TB), incubated at 37°C overnight, and plated onto brilliant green agar medium with novobiocin (BGAN). The TB was left at room temperature for 5 days and then used to inoculate fresh TB. The fresh TB was incubated at 37°C over night and plated onto BGAN medium. All the plates were incubated at 37°C over night and observed forSalmonella-like growth. Suspect colonies were further tested andSalmonella isolates were serotyped by the National Veterinary Services laboratory. Twenty-two of the 36 calves sampled harbouredS. typhimurium in their faeces, but no samples from cows were positive. NoSalmonella were isolated from the buffy coats, but 4 calves were shown to haveSalmonella in their nasal secretions. Extended enrichment of the faecal cultures in TB resulted in a significant increase inSalmonella isolations, although 2 samples were positive following the initial enrichment period and not after secondary enrichment. The typicalSalmonella isolate detected from this herd contained a transmissible R-plasmid encoding resistance to tetracycline, kanamycin, sulphisoxazole and ampicillin. This study confirmed that delayed secondary enrichment in TB is superior to primary enrichment for detection ofSalmonella from cattle.Abbreviations Ap ampicillin - BGAN brilliant green agar with novobiocin - Cm chloramphenicol - DSE delayed secondary enrichment - Gn gentamicin - Kn kanamycin - NA nalidixic acid - NADC National Animal Disease Center in Ames, Iowa - NVSL National Veterinary Services Laboratory in Ames, Iowa - PB polymyxin B - PE primary enrichment - Sm streptomycin - Su sulphisoxazole - TB tetrathionate broth - Tc tetracycline - TSI triple sugar iron agar     

Increasing prevalence of Coxiella burnetii seropositive Danish dairy cattle herds

A study based on bulk tank milk samples from 120 randomly selected dairy cattle herds was conducted to estimate the prevalence of Coxiella burnetii seropositive dairy herds, to describe the geographical distribution, and to identify risk factors. Using the CHEKIT Q-fever Antibody ELISA Test Kit (IDEXX), the study revealed a prevalence of 79.2% seropositive herds, 18.3% seronegative herds, and 2.5% serointermediate herds based on the instructions provided by the manufacturer. Multifactorial logistic regression showed statistically significant associations (P < 0.01) between C. burnetii seropositivity and increasing herd size (OR = 1.02 per cow increment) and increasing regional average number of cattle per dairy herd (OR = 1.02 per animal increment). Herds >150 cows had 17.9 times higher odds of testing positive compared to herds <80 cows. The regional average number of cattle herds per square kilometer was borderline significantly related to the occurrence of seropositive dairy herds (P = 0.06). The results indicate an increased prevalence of seropositive dairy herds since the previous survey in 2008 and an adverse impact of increasing herd size and cattle density on the risk of seropositivity.