Guidelines for the prevention of simian immunodeficiency virus infection in laboratory workers and animal handlers

The authors are members of a working group that formulated guidelines to minimize transmission of simian immunodeficiency virus (SIV) infection to man. Biosafety level (BSL) 2 standards are recommended for handling of clinical specimens and housing of SIV inoculated animals. Manipulation of SIV preparations may be performed in a BSL 2 facility with additional BSL 3 practices and equipment; for large volume or concentrated preparations of SIV BSL 3 containment is necessary. Written policies regarding management and testing of workers exposed to SIV are recommended.     

Preparing New World monkeys for laboratory research

New World monkeys represent an important but often poorly understood research resource. The relatively small size and low zoonotic risk of these animals make them appealing as research subjects in a number of areas. However, historic portrayal of many of these species as difficult to manage and handle is one of the factors that has limited their use. Basic guidelines are provided on management and handling approaches for the New World monkeys most commonly used in research: marmosets, squirrel monkeys, owl monkeys, and titi monkeys. Topics include transport and acclimation to a new facility, location changes within a facility, diet changes, removal from and return to social groups, capture and restraint, handling for anesthesia, postprocedural monitoring, and staff training.     

外科动物实验中猪的麻醉问题

猪作为重要的实验动物,在外科实验中运用传统的麻醉方法时易引起呼吸道阻塞而死亡,我们使用全麻插管技术,并辅以术前术后各种对症处理,成功地解决了手术中猪因呼吸道阻塞而死亡的问题。     

A playroom as novel swine enrichment

Pigs, intelligent and potentially destructive animals, can present unique challenges to laboratory animal caretakers trying to provide optimal housing and environmental enrichment. The authors developed an enrichment area in their facility for pigs that are neither preparing for nor recovering from surgery. They rotate pigs into the play area for periodic enrichment and socialization.     

Socialization strategies and disease transmission in captive colonies of nonhuman primates

In captive research environments for nonhuman primates (NHP), social housing strategies are often in conflict with protocols designed to minimize disease transmission. This is particularly true in breeding colonies, and is especially relevant when attempting to eliminate specific pathogens from a population of primates. Numerous strategies have been used to establish such specific pathogen free (SPF) breeding colonies (primarily of macaques), ranging from nursery rearing of neonates to single housing of socially reared yearlings to the rearing of infants in large social groups. All these strategies attempt to balance the effects of the chosen socialization strategy on parameters related to disease transmission, including the ultimate elimination of the target pathogens. Such strategies may affect the overall disease states of NHP breeding colonies through selective breeding processes. This can occur either by creating subpopulations of animals that do not have target diseases (SPF colonies), but may have other issues; or by creating situations in which the "best" animals are sold and the breeding colony is stocked with animals that may be more disease susceptible than those that were sold. The disease states of NHP research colonies also may be affected by selective utilization programs, in which animals removed from the breeding colony for health/behavior reasons, are preferentially chosen for use in scientific investigations. Such utilization criteria raise the question of whether ideal subjects are being chosen for use in research. Finally, captive primate colonies, where both socialization and disease states are intensely managed, may provide opportunities for those testing predictions from models of the interactions of socialization and disease transmission in the evolution of wild populations of NHP. This would be especially true for some extreme conditions of these disease ecology models, given the exceedingly high social densities and levels of pathogen control that exist in many captive nonhuman primate colonies.     

Perioperative socialization, care and monitoring of National Institutes of Health miniature swine undergoing ocular surgery and sampling of peripheral blood

Swine are a frequent species of choice for testing new surgical procedures and for transplantation studies. However, information concerning best practice to prepare pigs for surgery and postoperative treatment and monitoring is limited, despite a perception that preoperative socialization is beneficial. Therefore we examined the effect of preoperative visits by project personnel on compliance of 26 National Institutes of Health (NIH) minipigs subject to corneal transplantation. We briefly describe sedation and anaesthesia protocols developed for surgery and multiple postoperative interventions in order to facilitate interpretation of data relating to pig compliance. Preoperative visit variables and measures of preoperative socialization were correlated with postoperative outcome. Principal component analysis (PCA) of postoperative outcome variables identified a factor accounting for 53.5% of the variance that was significantly associated with two factors derived from PCA of preoperative factors (accounting, respectively, for 54.7% and 26.0% of the variance; P = 0.019 for the overall model, P = 0.041 and 0.040 for factors 1 and 2, respectively), such that more time spent with pigs before surgery and higher socialization scores were associated with less postoperative stress and difficulty of eye medication. Moreover, two of the preoperative visit variables, time spent with only one person in the pen and time spent with two or more people in the pen, contributed predominantly to PCA factors 1 and 2, respectively, indicating that they were fulfilling two qualitatively different requirements for socialization. We conclude that NIH minipigs are fully compliant with anaesthetic and postoperative experimental procedures provided they are well-socialized to project personnel before surgery.     

Review of trap-and-haul for managing Pacific salmonids (Oncorhynchus spp.) in impounded river systems

High-head dams are migration barriers for Pacific salmon Oncorhynchus spp. in many river systems and recovery measures for impacted stocks are limited. Trap-and-haul has been widely used in attempts to facilitate recovery but information from existing programs has not been synthesized to inform improvements to aid recovery of salmonids in systems with high-head dams. We reviewed 17 trap-and-haul programs regarding Pacific salmon to: (1) summarize information about facility design, operation and biological effects; (2) identify critical knowledge gaps; and (3) evaluate trap-and-haul as a current and future management tool. Existing programs are operated to address a range of management goals including restoring access to historical habitats, temporarily reducing exposure to dangerous in-river conditions, and reintroducing ecological processes upstream from dams. Information gathered from decades of operation on facility design criteria and fish handling protocols, and robust literature on fish collection and passage are available. While many aspects of trap-and-haul have been evaluated, effects on population productivity and sustainability remain poorly understood. Long-term and systematic studies of trap-and-haul outcomes are rare, and assessments can be confounded by concurrent management actions and broad ecological and climatic effects. Existing data suggest that performance and effectiveness vary among programs and over various time scales within programs. Although critical information gaps exist, trap-and-haul is an important management and conservation tool for providing Pacific salmonids access to historical habitats. Successful application of trap-and-haul programs requires long-term commitment and an adaptive management approach by dam owners and stakeholders, and careful planning of new programs.

     

Review: domestic animal forensic genetics – biological evidence,genetic markers,analytical approaches and challenges

This review highlights the importance of domestic animal genetic evidence sources, genetic testing, markers and analytical approaches as well as the challenges this field is facing in view of the de facto ‘gold standard’ human DNA identification. Because of the genetic similarity between humans and domestic animals, genetic analysis of domestic animal hair, saliva, urine, blood and other biological material has generated vital investigative leads that have been admitted into a variety of court proceedings, including criminal and civil litigation. Information on validated short tandem repeat, single nucleotide polymorphism and mitochondrial DNA markers and public access to genetic databases for forensic DNA analysis is becoming readily available. Although the fundamental aspects of animal forensic genetic testing may be reliable and acceptable, animal forensic testing still lacks the standardized testing protocols that human genetic profiling requires, probably because of the absence of monetary support from government agencies and the difficulty in promoting cooperation among competing laboratories. Moreover, there is a lack in consensus about how to best present the results and expert opinion to comply with court standards and bear judicial scrutiny. This has been the single most persistent challenge ever since the earliest use of domestic animal forensic genetic testing in a criminal case in the mid‐1990s. Crime laboratory accreditation ensures that genetic test results have the courts’ confidence. Because accreditation requires significant commitments of effort, time and resources, the vast majority of animal forensic genetic laboratories are not accredited nor are their analysts certified forensic examiners. The relevance of domestic animal forensic genetics in the criminal justice system is undeniable. However, further improvements are needed in a wide range of supporting resources, including standardized quality assurance and control protocols for sample handling, evidence testing, statistical analysis and reporting that meet the rules of scientific acceptance, reliability and human forensic identification standards.     

Nasal Wipes for Influenza A Virus Detection and Isolation from Swine

Surveillance for influenza A viruses in swine is critical to human and animal health because influenza A virus rapidly evolves in swine populations and new strains are continually emerging. Swine are able to be infected by diverse lineages of influenza A virus making them important hosts for the emergence and maintenance of novel influenza A virus strains. Sampling pigs in diverse settings such as commercial swine farms, agricultural fairs, and live animal markets is important to provide a comprehensive view of currently circulating IAV strains. The current gold-standard ante-mortem sampling technique (i.e. collection of nasal swabs) is labor intensive because it requires physical restraint of the pigs. Nasal wipes involve rubbing a piece of fabric across the snout of the pig with minimal to no restraint of the animal. The nasal wipe procedure is simple to perform and does not require personnel with professional veterinary or animal handling training. While slightly less sensitive than nasal swabs, virus detection and isolation rates are adequate to make nasal wipes a viable alternative for sampling individual pigs when low stress sampling methods are required. The proceeding protocol outlines the steps needed to collect a viable nasal wipe from an individual pig.     

New Instruments for High Throughput Receptor Binding Assays

Abstract

The TopCount^(R) Microplate Scintillation Counter and the Matrix 9600^(R) Direct Beta Counter are microplate compatible instruments developed to meet the needs of investigators using radioisotope assays adapted for very high throughput. This paper describes these instruments and their application to receptor binding assays. When combined with the appropriate sample handling equipment and filter media, use of these multi-detector instruments improves sample handling efficiency and shortens overall counting time. The assay protocols including filtration through glass fiber mats and membrane filters have been investigated. Results obtained from these new instruments are compared to standard techniques using conventional liquid scintillation and gamma counting.     

Distribution of biological databases over lowbandwidth networks

Databases are integral part of bioinformatics and need to be accessed most frequently, thus downloading and updating them on a regular basis is very critical. The establishment of bioinformatics research facility is a challenge for developing countries as they suffer from inherent low-bandwidth and unreliable internet connections. Therefore, the identification of techniques supporting download and automatic synchronization of large biological database at low bandwidth is of utmost importance. In current study, two protocols (FTP and Bit Torrent) were evaluated and the utility of a BitTorren based peer-to-peer (btP2P) file distribution model for automatic synchronization and distribution of large dataset at our facility in Pakistan have been discussed.     

A proteomics approach to identifying fish cell lines

Fish cell lines are relatively easy to culture and most have simple growth requirements that make cross contamination a potential problem. Cell line contamination is not an uncommon incident in laboratories handling more than one cell line and many reports have been made on cross contamination of mammalian cell lines. Although problems of misidentification and cross-contamination of fish cell lines have rarely been reported, these are issues of concern for cell culturists that can make scientific results and their reproducibility unreliable. Proper identification of cell lines is thus crucial and protocols for routine and rapid screening are preferred. Cytogenetic evaluation, DNA fingerprinting, microsatellite analysis and PCR methods have been published for inter-species identification of many cell lines, but discerning intra-species contamination has been challenging. More complex DNA fingerprinting and hybridization techniques coupled with isoenzyme analysis have been developed to discriminate intra-species contamination, however, these require complex and time consuming procedures to enable cell identification thus are difficult to apply for routine use. A simple proteomic approach has been made to identify several fish cell lines derived from tissues of the same or differing species. Protein expression signatures (PES) of the evaluated fish cell lines have been developed using 2-DE and image analysis. A higher degree of concordance was seen among cell lines derived from rainbow trout, than from other fish species. Similar concordance was seen in cells derived from the same tissues than from other tissues within the same species. These profiles have been saved in an electronic databank and could be made available to be used for discerning the origins of the various cell lines evaluated. This proteomic approach could thus serve as an additional, valuable and reliable technique for the identification of fish cell lines.     

Applications and optimization of immunization procedures

Classical immunization protocols have produced an antibody-based humoral response that is very effective against susceptible infectious diseases. Immunization introduces an external substance to induce the host immune system to respond specifically. Typically an antigen is used, but DNA, or a primed, pre-existing leukocyte or antigen-presenting cell, can also be used. Immunization is currently being used or investigated for the prevention and treatment of infectious diseases, cancer, addictions, allergies, pregnancy, and autoimmune diseases. It is also being used to produce biologically active materials such as polyclonal and monoclonal antibodies, antivenins, and anti-toxins for treating a wide range of conditions. Animals have been integral to the development of immunization techniques, as producers of toxoids and antitoxins, as models (e.g., to validate materials and protocols used for immunization, to understand the impact of immunization itself on the immune system, and to help investigators devise methods for determining the efficacy of vaccines) and as beneficiaries themselves of vaccines and antitoxins. The choice of immunization protocols is complex, and results may be affected by many factors such as dose and concentration of antigen, choice of adjuvants, time between inoculation and response measurement, and method of detection. The immune system responses to an antigen are also complex and continue to develop with advancing age. Anatomical, physiological, and immune system differences between species influence responses to immunization, as do the purity and presentation of the antigens and adjuvants. When directly comparing results, animals should be sourced from the same supplier. This review highlights the many uses of immunization techniques and introduces important considerations for the choice of protocols and animal models.     

Institutional responsibilities in contamination control in research animals and occupational health and safety for animal handlers

The mechanisms for controlling microbial contamination in research animals are similar to those for preventing exposure among animal handlers to naturally occurring pathogens, research-related biohazards, or animal allergens. Research and resource preservation are the primary goals of each approach, and an appropriate assessment of risk is their foundation. The identification of potential risks enables the implementation of relevant risk management or control measures. This article summarizes the components of an occupational health and safety program for animal handlers, including screening, training, work practices, effective use of engineering controls, selection and use of personal protective equipment, and emergency response protocols. The features of a risk assessment and risk management program and the level of interaction and training required to implement and sustain the program correlate well with programs designed to control microbial contamination in laboratory animals. This article includes an explanation of the five Ps of risk assessment and risk management. Pathogens and the proposed experimental procedures account for the first two Ps; the other three focus on training and awareness of the personnel involved in the experiment, protective equipment and work practices, and factors associated with the place (or facility) where the research will be conducted. Animal handlers comprehension and knowledge determine the success of any containment program, and so this review also includes a discussion of critical teaching points for animal handlers and of the importance of evaluating personnel to verify their proficiency and competence in required protocols.     

Phosphorothioate-based ligase-independent gene cloning (PLICing): An enzyme-free and sequence-independent cloning method

Many ligase-independent cloning methods have been developed to overcome problems of standard restriction cloning such as low transformation efficiency and high background of vector with no insert. Most of these methods are still enzyme based, require time-consuming incubation and multiple purification steps, and/or might have a low robustness in handling. Thus, with the aim to establish a robust enzyme/ligase-free method, we developed the phosphorothioate-based ligase-independent gene cloning (PLICing) method, which is based on a chemical cleavage reaction of phosphorothioate bonds in an iodine/ethanol solution. After optimization of polymerase chain reaction (PCR) and DNA cleavage conditions, PLICing performs competitively with all commercialized methods in terms of handling and transformation efficiency. In addition, PLICing is absolutely sequence independent and surpasses other concepts regarding cloning efficiency given that none of the 240 analyzed clones showed any religation event for three different model genes. A developed fast PLICing protocol does not require any purification step and can be completed within 10 min. Due to its robustness, reliability, and simplicity, PLICing should prove to be a true alternative to other well-established cloning techniques.     

Quality standards in proteomics research facilities: Common standards and quality procedures are essential for proteomics facilities and their users

Proteomics research infrastructures and core facilities within the Core for Life alliance advocate for community policies for quality control to ensure high standards in proteomics services.

Core facilities and research infrastructures have become an essential part of the scientific ecosystem. In the field of proteomics, national and international networks and research platforms have been established during the past decade that are supposed to set standards for high‐quality services, promote an exchange of professional information, and enable access to cutting‐edge, specialized proteomics technologies. Either centralized or distributed, these national and international proteomics infrastructures and technology platforms are generating massive amounts of data for the research community, and support a broad range of translational, computational and multi‐omics initiatives and basic research projects.By delegating part of their work to these services, researchers expect that the core facility adjusts their analytical protocols appropriately for their project to acquire data conforming best research practice of the scientific community. The implementation of quality assessment measures and commonly accepted quality controls in data generation is therefore crucially important for proteomics research infrastructures and the scientists who rely on them.However, current quality control and quality assessment procedures in proteomics core facilities and research infrastructures are a motley collection of protocols, standards, reference compounds and software tools. Proteomics relies on a customized multi‐step workflow typically consisting of sample preparation, data acquisition and data processing, and the implementation of each step differs among facilities. For example, sample preparation involves enzymatic digestion of the proteins, which can be performed in‐solution, in‐gel, or on‐beads, with often different proteolytic enzymes, chemicals, and conditions among laboratories. Data acquisition protocols are often customized to the particular instrument set up, and the acquired spectra and chromatograms are processed by different software tools provided by equipment vendors, third parties or developed in‐house.

…current quality control and quality assessment procedures in proteomics core facilities and research infrastructures are a motley collection of protocols, standards, reference compounds and software tools.

Moreover, core facilities implement their own guidelines to monitor the performance and quality of the entire workflow, typically utilizing different commercially available standards such as pre‐digested cell lysates, recombinant proteins, protein mixtures, or isotopically labeled peptides. Currently, there is no clear consensus on if, when and how to perform quality control checks. There is even less quality control in walk‐in facilities, where the staff is only responsible for correct usage of the instruments and users select and execute the analytical workflow themselves. It is not surprising therefore that instrument stability and robustness of the applied analytical approach are often unclear, which compromises analytical rigor.     

Current good manufacturing practice in plant automation of biological production processes

The production of biologicals is subject to strict governmental regulations. These are drawn up in current good manufacturing practices (cGMP), a.o. by the U.S. Food and Drug Administration. To implement cGMP in a production facility, plant automation becomes an essential tool. For this purpose Manufacturing Execution Systems (MES) have been developed that control all operations inside a production facility. The introduction of these recipe-driven control systems that follow ISA S88 standards for batch processes has made it possible to implement cGMP regulations in the control strategy of biological production processes. Next to this, an MES offers additional features such as stock management, planning and routing tools, process-dependent control, implementation of software sensors and predictive models, application of historical data and on-line statistical techniques for trend analysis and detection of instrumentation failures. This paper focuses on the development of new production strategies in which cGMP guidelines are an essential part.     

Should I Stay or Should I go? The Influence of Handling by Researchers on Den use in an Arboreal Nocturnal Rodent

Understanding how animals respond to disturbance by investigators is essential for a fair assessment of the presence of bias in routinely used research protocols. It is also an essential prerequisite for anyone interested in animal welfare and ethically sound research. Here, we utilize an automatic logging system to monitor nest box use by PIT‐tagged edible dormice, Glis glis, after standard handling procedures applied during a regular nest‐box monitoring programme. The edible dormouse is an arboreal nocturnal rodent that relies on tree hollows as daytime den sites. We assessed the effect of disturbance on dormice in two ways: whether it affected the decision of an individual to stay in the same den site for a subsequent day and whether it affected the timing of the individual's nocturnal emergence from the den site. We found handling had a strong negative effect on short‐term den use. In addition, females and sexually active individuals were more likely to spend the following day in the nest box. Individuals that had left the den site after our handling returned to them after an average of 4 d. Handling did not have a significant effect on the period of absence, but reproductively active animals returned on average after 3 d, while reproductively quiescent animals returned after more than 5 d. Manipulation did not have a significant effect on the initiation of nocturnal activity. Our study suggests that disturbance by investigators may modify certain aspects of animal behaviour, but this effect is likely to be short term and does not appear to impair the efficacy of routinely practiced capture‐mark‐recapture field protocols.