Distribution of Bacteria in Feces of Swine

A new technique is described for evaluating bacterial cell distribution in fecal samples. Spatial relationships of cells within an area rather than number of cells per unit volume or weight are measured by this technique. Measurements of cell distribution by this method indicated that bacteria occurred in freshly voided swine feces as pure, discrete colonies rather than as single cells distributed randomly or uniformly throughout the sample.     

Convenient Sampling of Air Bacteria in Operating Rooms

A convenient arrangement for sampling air bacteria in operating rooms with a slit sampler (the Fort Detrick sampler) is described. Its purpose is to contribute as far as possible to the convenience of the surgical staff and thereby to the safety of the patient. It has the advantages of recording minute-to-minute changes in bacterial air count; it is unobtrusive and yet can be continually observed by a technician; it is not noisy and avoids the dangers of explosion and static electricity; it is inexpensive, and parts are easily replaced; and finally it provides a means of keeping permanent photographic records of bacterial counts. Results of a preliminary trial of this method appeared to be satisfactory.     

Butyrate-Producing Bacteria,Including Mucin Degraders,from the Swine Intestinal Tract

To identify bacteria with potential for influencing gut health, 980 anaerobes were cultured from the swine intestinal tract and analyzed for butyrate production. Fifteen isolates in the order Clostridiales produced butyrate and had butyryl coenzyme A (CoA):acetate CoA transferase activity. Three of the isolates grew on mucin, suggesting an intimate association with host intestinal mucosa.     

Medium for the Enumeration and Isolation of Bacteria from a Swine Waste Digester

A habitat-simulating medium was developed for the enumeration and isolation of bacteria from a swine waste digester. A roll tube medium with growth factors for strict anaerobes from previously studied anaerobic ecosystems was used to evaluate the effects of deletion, addition, or level of digester fluid, digester fluid treated with acid or base, rumen fluid, fecal extract, anaerobic pit extract, tissue extract, carbohydrates, peptones, short-chain fatty acids, minerals, vitamins, N and P sources, reducing and solidifying agents, buffers, and gases on colony counts. Decreasing the agar concentration from 2.5 to 1.0% increased the counts twofold. Blending increased the counts 1.7-fold. With a medium (174) containing digester fluid, peptones, minerals, cysteine, sodium carbonate, and agar, colony counts were 60% of the microscopic count and improved yields 2.5 to 20 times those obtained with media previously used for digesters or developed for other anaerobic ecosystems. Colony counts continued to increase for up to 4 weeks of incubation. Medium 174 permits the enumeration of total, methanogenic, and, with deletion of reducing agent, aerotolerant bacteria. The results suggest that the predominant bacteria grow slowly and have requirements different from those of bacteria from other ecosystems.     

Medium for Enhanced Growth of Bacteria from a Swine Manure Digester

Most bacterial strains isolated from a swine manure digester grew sufficiently to permit transfer of cultures, but not characterization. Substrates, crude extracts, growth factors, and electron acceptors were evaluated for growth promotion. The growth of all but one group of the isolates was substantially increased with a medium containing glucose, cellobiose, soluble starch, pyruvate, peptone, yeast extract, minerals, volatile acids, vitamins, hemin plus vitamins K₁ and K₃, sodium bicarbonate, cysteine, and digester fluid. The strains require both known and unknown factors (in crude extracts) for maximal growth.     

重症监护病房常见非发酵菌分布及耐药性分析

分析医院重症监护病房非发酵菌感染的耐药情况,以指导临床合理使用抗菌药物。回顾性分析2009~2010年医院自重症监护病房分离的非发酵菌,对其检出率及药敏结果进行统计分析。共检出96株非发酵菌,检出率为19.79%,分离率居前4位的依次为铜绿假单胞菌（46.88%）、鲍氏不动杆菌（21.88%）、嗜麦芽寡养单胞菌（15.63%）、洋葱伯克霍尔德菌（10.42%）;4种常见的非发酵菌对常用的抗菌药物耐药性均较高,头孢哌酮/舒巴坦等含酶抑制剂的复合型抗菌药物对非发酵菌有较高的敏感性。医院重症监护病房非发酵菌检出率高且耐药性强,应加强临床细菌学的检测,按照药敏试验结果合理用药。     

Oligophilic Bacteria as Tools To Monitor Aseptic Pharmaceutical Production Units

The bacterial loads of air, surfaces, and personnel in clean rooms are routinely monitored using a set of standard media. Bacteria that can grow on these media are a tiny fraction of the total numbers in any environment. A substantial proportion of bacteria long thought to be unculturable were recently shown to be oligophilic. Oligophile counts in clean rooms in our studies exceeded the standard plate counts by up to 2 orders of magnitude. They responded to disinfection routines in ways similar to the responses of conventional bacteria. We suggest that oligophiles are better tools than conventional bacteria for environmental monitoring in aseptic pharmaceutical production units.     

Isolation and Identification of Hyper-Ammonia Producing Bacteria from Swine Manure Storage Pits

Storage of swine manure is associated with the microbiological production of a variety of odorous compounds, including ammonia, organic acids, and alcohols, phenolics, and sulfides. Until recently, little was known about the microorganisms responsible for their production. Results from our laboratory have demonstrated that the predominant microbial populations of stored swine manure are anaerobic, low (G + C), Gram-positive bacteria. However, studies on pure cultures isolated from manure have found few microorganisms that produce appreciable ammonia concentrations. Therefore, selective and enrichment techniques were employed to isolate ammonia-producing bacteria from stored swine manure by using media containing peptone and amino acids as carbon and energy sources. We now report on the isolation of 40 bacterial cultures, a number of which are capable of producing at least 40 mM ammonia in peptone-amino acid medium, concentrations similar to those produced by hyper-ammonia producing (HAP) bacteria isolated from the rumen of cattle. The manure HAP isolates are phylogenetically distinct from the ruminal isolates and may prove to be intimately involved in the production of ammonia during storage of swine manure.     

Impact of an Aerobic Thermophilic Sequencing Batch Reactor on Antibiotic-Resistant Anaerobic Bacteria in Swine Waste

The introduction of antibiotics to animal feed has contributed to the selection of antibiotic-resistant bacteria in concentrated animal feeding operations. The aim of this work was to characterize the impact of an aerobic thermophilic biotreatment on anaerobic antibiotic-resistant bacteria in swine waste. Despite 162- to 6,166-fold reduction in antibiotic-resistant populations enumerated in the swine waste at 25°C and 37°C, resistant populations remained significant (10⁴ to 10⁵ most probable number per milliliter) in the treated swine waste. Five resistance genes were detected before [tet(LMOS) erm(B)], and six resistance genes were detected after [tet(LMOSY) erm(B)] biotreatment. However, the biotreatment decreased the frequency of detection of resistance genes by 57%. Analysis by denaturing gradient gel electrophoresis of polymerase chain reaction-amplified 16 S ribosomal DNA (rDNA) fragments showed that the biotreatment reduced the bacterial diversity of resistant populations enumerated at 37°C. Cloning and sequencing of the 16 S rDNA of these populations revealed that most clones in the treated swine waste were closely similar to some of the clones retrieved from the untreated swine waste. This study revealed that the aerobic thermophilic biotreatment developed in our laboratory does not prevent the introduction of facultatively anaerobic antibiotic-resistant bacteria and their resistance genes into agricultural ecosystems. Horizontal transfer of ecologically advantageous genes within microbial communities are likely to prevent thermophilic biotreatments from completely eliminating antibiotic-resistant bacteria and their resistance genes in animal wastes.     

Detection and Isolation of Swine Influenza A Virus in Spiked Oral Fluid and Samples from Individually Housed,Experimentally Infected Pigs: Potential Role of Porcine Oral Fluid in Active Influenza A Virus Surveillance in Swine

Background

The lack of seasonality of swine influenza A virus (swIAV) in combination with the capacity of swine to harbor a large number of co-circulating IAV lineages, resulting in the risk for the emergence of influenza viruses with pandemic potential, stress the importance of swIAV surveillance. To date, active surveillance of swIAV worldwide is barely done because of the short detection period in nasal swab samples. Therefore, more sensitive diagnostic methods to monitor circulating virus strains are requisite.

Methods

qRT-PCR and virus isolations were performed on oral fluid and nasal swabs collected from individually housed pigs that were infected sequentially with H1N1 and H3N2 swIAV strains. The same methods were also applied to oral fluid samples spiked with H1N1 to study the influence of conservation time and temperature on swIAV infectivity and detectability in porcine oral fluid.

Results

All swIAV infected animals were found qRT-PCR positive in both nasal swabs and oral fluid. However, swIAV could be detected for a longer period in oral fluid than in nasal swabs. Despite the high detectability of swIAV in oral fluid, virus isolation from oral fluid collected from infected pigs was rare. These results are supported by laboratory studies showing that the PCR detectability of swIAV remains unaltered during a 24 h incubation period in oral fluid, while swIAV infectivity drops dramatically immediately upon contact with oral fluid (3 log titer reduction) and gets lost after 24 h conservation in oral fluid at ambient temperature.

Conclusions

Our data indicate that porcine oral fluid has the potential to replace nasal swabs for molecular diagnostic purposes. The difficulty to isolate swIAV from oral fluid could pose a drawback for its use in active surveillance programs.     

Characterization of Phototrophic Purple Nonsulfur Bacteria Forming Colored Microbial Mats in a Swine Wastewater Ditch

The community structure of pink-colored microbial mats naturally occurring in a swine wastewater ditch was studied by culture-independent biomarker and molecular methods as well as by conventional cultivation methods. The wastewater in the ditch contained acetate and propionate as the major carbon nutrients. Thin-section electron microscopy revealed that the microbial mats were dominated by rod-shaped cells containing intracytoplasmic membranes of the lamellar type. Smaller numbers of oval cells with vesicular internal membranes were also found. Spectroscopic analyses of the cell extract from the biomats showed the presence of bacteriochlorophyll a and carotenoids of the spirilloxanthin series. Ubiquinone-10 was detected as the major quinone. A clone library of the photosynthetic gene, pufM, constructed from the bulk DNA of the biomats showed that all of the clones were derived from members of the genera Rhodobacter and Rhodopseudomonas. The dominant phototrophic bacteria from the microbial mats were isolated by cultivation methods and identified as being of the genera Rhodobacter and Rhodopseudomonas by studying 16S rRNA and pufM gene sequence information. Experiments of oxygen uptake with lower fatty acids revealed that the freshly collected microbial mats and the Rhodopseudomonas isolates had a wider spectrum of carbon utilization and a higher affinity for acetate than did the Rhodobacter isolates. These results demonstrate that the microbial mats were dominated by the purple nonsulfur bacteria of the genera Rhodobacter and Rhodopseudomonas, and the bioavailability of lower fatty acids in wastewater is a key factor allowing the formation of visible microbial mats with these phototrophs.     

Airborne Detection and Quantification of Swine Influenza A Virus in Air Samples Collected Inside,Outside and Downwind from Swine Barns

Airborne transmission of influenza A virus (IAV) in swine is speculated to be an important route of virus dissemination, but data are scarce. This study attempted to detect and quantify airborne IAV by virus isolation and RRT-PCR in air samples collected under field conditions. This was accomplished by collecting air samples from four acutely infected pig farms and locating air samplers inside the barns, at the external exhaust fans and downwind from the farms at distances up to 2.1 km. IAV was detected in air samples collected in 3 out of 4 farms included in the study. Isolation of IAV was possible from air samples collected inside the barn at two of the farms and in one farm from the exhausted air. Between 13% and 100% of samples collected inside the barns tested RRT-PCR positive with an average viral load of 3.20E+05 IAV RNA copies/m³ of air. Percentage of exhaust positive air samples also ranged between 13% and 100% with an average viral load of 1.79E+04 RNA copies/m³ of air. Influenza virus RNA was detected in air samples collected between 1.5 and 2.1 Km away from the farms with viral levels significantly lower at 4.65E+03 RNA copies/m³. H1N1, H1N2 and H3N2 subtypes were detected in the air samples and the hemagglutinin gene sequences identified in the swine samples matched those in aerosols providing evidence that the viruses detected in the aerosols originated from the pigs in the farms under study. Overall our results indicate that pigs can be a source of IAV infectious aerosols and that these aerosols can be exhausted from pig barns and be transported downwind. The results from this study provide evidence of the risk of aerosol transmission in pigs under field conditions.     

Fate of Chlortetracycline- and Tylosin-Resistant Bacteria in an Aerobic Thermophilic Sequencing Batch Reactor Treating Swine Waste

Antibiotics have been added to animal feed for decades. Consequently, food animals and their wastes constitute a reservoir of antibiotic-resistant bacteria. The objective of this work was to characterize the impact of an aerobic thermophilic biotreatment on aerobic, antibiotic-resistant bacteria in swine waste. The proportion of tylosin- and chlortetracycline-resistant bacteria grown at 25°C, 37°C, and 60°C decreased after treatment, but they were still abundant (10² to 10⁸ most probable number ml⁻¹) in the treated swine waste. The presence of 14 genes conferring resistance to tylosin and chlortetracycline was assessed by polymerase chain reaction in bacterial populations grown at 25°C, 37°C, and 60°C, with or without antibiotics. In 22 cases, genes were detected before but not after treatment. The overall gene diversity was wider before [tet(BLMOSY), erm(AB)] than after [tet(LMOS), erm(B)] treatment. Analysis by denaturing gradient gel electrophoresis of amplified 16S ribosomal DNA (rDNA) fragments generally showed a reduction of the bacterial diversity, except for total populations grown at 60°C and for tylosin-resistant populations grown at 37°C. The latter were further investigated by cloning and sequencing their 16S rDNA. Phylotypes found before treatment were all closely related to Enterococcus hirae, whereas six different phylotypes, related to Pseudomonas, Alcaligenes, and Pusillimonas, were found after treatment. This work demonstrated that the aerobic thermophilic biotreatment cannot be considered as a means for preventing the dissemination of aerobic antibiotic-resistant bacteria and their resistance genes to the environment. However, since pathogens do not survive the biotreatment, the effluent does not represent an immediate threat to animal or human health.     

Elimination Behavior of Shelter Dogs Housed in Double Compartment Kennels

For animals in confinement housing the housing structure has tremendous potential to impact well being. Dogs in animal shelters are often housed in one of two types of confinement housing – single kennels and rooms or double compartment kennels and rooms most often separated by a guillotine door. This study examines the effect of housing on the location of elimination behavior in dogs housed in double compartment kennels were the majority of the dogs were walked daily. One side of the kennel contained the food, water and bed and the other side was empty and available except during cleaning time. Location of urination and defecation was observed daily for 579 dogs housed in indoor double compartment kennels for a total of 4440 days of observation. There were 1856 days (41.9%) when no elimination was noted in the kennel. Feces, urine or both were observed in the kennel on 2584 days (58.1%). When elimination occurred in the kennel the probability of fecal elimination on the opposite side of the bed/food/water was 72.5% (95% CI 69.05% to 75.69%). The probability of urination on the opposite side of the bed/food/water was 77.4% (95% CI 74.33% to 80.07%). This study demonstrates the strong preference of dogs to eliminate away from the area where they eat, drink and sleep. Double compartment housing not only allows this – it allows staff the ability to provide safe, efficient, humane daily care and confers the added benefits of reducing risks for disease transmission for the individual dog as well as the population.     

Relative Humidity and the Killing of Bacteria: The Survival of Damp Serratia marcescens in Air

The viability of washed moist cells of Serratia marcescens after storage has been measured in relation to variations in the prior treatment of the cells and in conditions of storage. The factors considered were: (i) water content during storage; (ii) method of arriving at water content (partial drying in vacuum or freeze-drying and addition of water); (iii) presence or absence of air during storage.

Increasingly rapid decay occurs as the water content at which the cells are stored is diminished from above 90% to 20 or 30% (“critical” water content). It occurs in presence or absence of air and it occurs whether the final water content is approached by removal of water from wet cells or by addition of water to freeze-dried cells.

The rate of decay during storage at 20 to 30% water is somewhat diminished by the presence of air (“protective” effect of air).

As the water content is further reduced to less than 10%, the stability of cells stored in a vacuum approaches that of wet cells. In presence of air the reverse is true: the stability decreases until at less than 1% water, the decay rate is about as great as at the “critical” water content (“toxic” effect of air).

Particularly rapid decay of S. marcescens at the “critical” water content has escaped attention in aerosol studies because accurate control of relative humidity (RH) in this region, RH 94 to 99%, is virtually impossible in such studies. On the other hand, values of decay rates referred to measured water contents are quite unreliable in the 20 to 80% RH zone because the corresponding variation of water content is too small to measure reliably. Thus data of the kind reported in this paper cannot be directly compared to the published results of studies of air-borne bacteria, although they are relevant to the practical question of air-borne infection in humid atmospheres.

     

Survival of Strains of Soft Rot Coliform Bacteria on Microthreads Exposed in the Laboratory and in the Open Air

Survival of 5 strains of the plant pathogen, Erwinia carotovora var. atroseptica and 3 strains of the plant pathogen E. carotovora var. carotovora , grown in a liquid tryptone medium and held as 'captive'aerosols on gossamer microthreads, was determined under different atmospheric conditions in a controlled environment room and in the open air. Although these bacteria lost viability more quickly than a robust reference strain of Escherichia coli , sufficient numbers survived for 15 min or more to indicate that airborne spread of viable propagules could take place, especially under cool humid atmospheric conditions. Cells of one strain of each organism extracted from rotted potato tuber tissue were shown to behave rather like those cultured in the tryptone medium.     

The Impact of Cognitive Testing on the Welfare of Group Housed Primates

Providing cognitive challenges to zoo-housed animals may provide enriching effects and subsequently enhance their welfare. Primates may benefit most from such challenges as they often face complex problems in their natural environment and can be observed to seek problem solving opportunities in captivity. However, the extent to which welfare benefits can be achieved through programmes developed primarily for cognitive research is unknown. We tested the impact of voluntary participation cognitive testing on the welfare of a socially housed group of crested macaques (Macaca nigra) at the Macaque Study Centre (Marwell Zoo). First, we compared the rate of self-directed and social behaviours on testing and non-testing days, and between conditions within testing days. Minimal differences in behaviour were found when comparing testing and non-testing days, suggesting that there was no negative impact on welfare as a result of cognitive testing. Lipsmacking behaviours were found to increase and aggressive interaction was found to decrease in the group as a result of testing. Second, social network analysis was used to assess the effect of testing on associations and interactions between individuals. The social networks showed that testing subjects increased their association with others during testing days. One interpretation of this finding could be that providing socially housed primates with an opportunity for individuals to separate from the group for short periods could help mimic natural patterns of sub-group formation and reunion in captivity. The findings suggest, therefore, that the welfare of captive primates can be improved through the use of cognitive testing in zoo environments.