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Zebrafish Health Monitoring
Speaker: J-P Mocho, Daniovet, UK    
Following the morbidity and mortality in housing systems is an essential tool to monitor the health of a colony. It should be completed with a definition of the health status, i.e., the presence or absence of specific pathogens. Some unwelcome microbes can impair staff health for example. Laboratory Danio rerio (zebrafish) rarely suffer from severe infections, but such contagious diseases can induce a significant peak of mortality; some less severe infections are common, and are likely to interfere with the quality and reproducibility of experiments. Therefore, it is necessary to define the relevant microbes to monitor according to their pathogenicity and prevalence. To perform the screening, the use of sentinels is a valuable option when it is performed in optimized conditions. The cadavers can be screened thanks to fresh mounts or histopathology. PCR testing is also available and allows the identification of pathogens from animal as well as environmental samples. Indeed, screening the environment allows a more sensitive detection of some relevant pathogens – and it is a good example of applying the 3Rs.
Identifying and Managing Critical Control Points to Reduce the Risk of Adventitious Rodent Infections
Speaker: Guy B. Mulder, Charles River Laboratories, Wilmington, USA
A successful biosecurity program encompasses all measures necessary to prevent, contain, and eradicate adventitious (unwanted) infectious agents from entering and circulating within a vivarium. To be successful, such a program must be designed to identify all possible infectious risks, i.e., critical control points, and implement effective processes to mitigate those risks. This presentation will review the critical control points that are commonly found in a rodent vivarium as well provide recommendations on how to effectively mitigate them.
Interceptor: “Interviewing” Air Exhaust Particles by PCR to Understand the Microbiological Status of Laboratory Rodents
Speaker: Gianpaolo Milite, Tecniplast, Italy
There at least two reasons behind the need for improvements in the approach used for the health (microbiological) monitoring of rodents in research. The first is that sentinel animals to soiled bedding do not detect many infectious agents; the second pertains to the actually more diffused rodents’ housing system, IVCs, and its ability to prevent aerogenic pathogen transmission and thereby providing bio-exclusion and bio-containment limiting the spread of infections.
Dust particles actively removed from each individually-ventilated cage are collected and concentrated in the exhaust filtering area of the hair handling units, representing an excellent potentially contaminated substrate for PCR investigation.
The development of a filtering device in closed system to avoid contamination of samples, positioned in a way to maximize particles collection (Interceptor), is a technical answer to the limiting factors above mentioned for efficient microbiological monitoring of rodent colonies in IVCs. Validated at Helmholtz Zentrum in Munich in collaboration with Dr. M. Brielmeier and later on by international laboratories, it can be used following different protocols or Decisional Algorithms.
Practical Applications in a Research Facility
Speaker: Julie Watson, Associate Professor, Dept. of Molecular & Comparative Pathobiology – Johns Hopkins School of Medicine

Recent advances in diagnostic methods have prompted many institutions, including our own, to consider radically changing the way we conduct health monitoring in our rodent facilities. The advent of exhaust air dust (EAD) testing by PCR has allowed us to identify colony organisms that were poorly detected by sentinels, and offers the enticing possibility that sentinels might be going the way of the dodo (a phrase shamelessly stolen from Ken Henderson). However, the question remains as to whether EAD testing is effective for all commonly excluded organisms or is instead subject to the vagaries of different rack systems, different bedding, and even different diagnostic laboratories. EAD testing also brings its own challenges, in particular an increase in false positives caused by ‘legacy’ DNA remaining in racks from prior infections. This talk will describe some of the successes and failures encountered in implementing an EAD health surveillance system and methods used to overcome problems. Suggestions will also be made for refinements to other health surveillance systems where EAD testing is not applicable.


Microbiologic Quality Control of Rodent Research Models: Biosecurity and Surveillance
Speaker: Bill Shek, Senior Scientific Director – Charles River

It is widely accepted that laboratory animals for modern biomedical research need to be specific pathogen free (SPF), i.e., free from infections with a limited list of pathogens shown to cause disease or otherwise interfere with research. The purpose of this presentation is to provide veterinarians and vivarium managers with an overview of the key components of microbiologic quality control (QC) to preserve the SPF status of research colonies including biosecurity and routine diagnostic testing, commonly called health monitoring (HM).


Effective biosecurity is based on mitigating the risks associated with the key animal and indirect sources of infection. Its main components for rodents are rederivation (by hysterectomy and cross-fostering, or embryo transfer) to eliminate adventitious agents, and maintenance of animals behind room- to cage-level barrier systems to exclude pathogens. For HM, the traditional diagnostic methodologies in use for over half a century include direct gross and microscopic examinations of animal specimens for parasites and pathology, microbiology consisting of cultural isolation and phenotypic identification of primary and opportunistically pathogenic bacteria and fungi, and serology (i.e., immunoassays) for specific antibodies chiefly to viruses. The newest methodology, molecular diagnostics by the polymerase chain reaction (PCR) was introduced in the mid-1990s; since then, its use in HM has grown, in recent years dramatically, for reasons to be discussed. Although advances in microbiologic QC have essentially eliminated once common pathogens such as Sendai virus, contaminations with environmentally stable and highly contagious enterotropic pathogens continue to occur. Moreover, these and recently discovered pathogens are prevalent in the genetically engineered mouse models that compose a rapidly growing percentage of research animals. The continued occurrence of adventitious infections and discovery of pathogens underscore the importance of HM results that accurately represent the pathogen status of research animals, but also highlight the limitations of HM and the benefits of rederivation to eliminate and prevent the dissemination of undiscovered pathogens. To avoid needless disruption to research, it is essential before taking corrective action to substantiate new positive findings by repeat testing of both the original and additional samples, utilizing available complementary diagnostic methodologies (e.g., serology and PCR). Corrective action plans should comprise communication with users and steps to contain, eradicate, investigate and mitigate a recurrence of the outbreak.



New Frontiers in NonHuman Primate Diagnostics
Speaker: Rajeev Dhawan, Director, Bioassay Services – Charles River

Routine health monitoring of laboratory nonhuman primates (NHPs) is performed by serology, PCR and bacteriology. Historically, serology using whole virus lysate ELISAs have been the main screening tool for detecting antibodies against the NIH recommended SPF infectious agents, mainly retroviruses (SIV, SRV and STLV) and B-virus. Emerging technologies are leading the way to improve routine diagnostic testing by providing faster, reliable results. These include use of recombinant antigens and multiplex Luminex® technology (MFIA) in which all assays for infectious agents (and internal controls) can be performed in a single well. Multiplexing reduces the need for large serum volume, allowing the use of a new sampling technique, HemaTIP™ for routine serosurveillance. HemaTIP™ requires only 20 µL of whole blood and NHPs need not be anesthetized during collection. Similarly, use of MALDI-TOF leads to cheaper and quicker identification of bacterial isolates in comparison to biochemical and PCR identification. In the past few years, Chagas and flavivirus infections have become of concern in outdoor NHP colonies, especially in southern USA. Assay development for these agents using these new technologies, as well as their prevalence data in lab NHPs, will be presented. In addition, results analysis and interpretation of various diagnostic tests will be discussed.


Quarantine Procedures for Laboratory Zebrafish
Speaker: Christian Lawrence, Aquatic Resources Program Manager – Boston Children’s Hospital


Pathogen control is a critical concern for laboratory zebrafish populations, given the threats posed by both outbreaks of clinical disease and from unplanned experimental variation arising from subclinical infections. Health management practices for zebrafish are in a state of flux, rapidly transitioning from antiquated/unproven protocols to more data-driven and performance-based approaches. A core element of an effective strategy is the ability to establish control over incoming materials that have the potential to transmit infectious agents to an existing population. This presentation will provide an overview of common “quarantine” procedures currently employed in the field to deal with this problem, as well as an introduction to concepts and emerging data that can be used to develop improved methodology going forward.



Creating a Health Management Program for Zebrafish Establishments
Speaker: Nick King, Director of Operations – Fish Vet Group

Conditions within aquatic systems favor the proliferation of many disease-causing organisms, including primary and opportunistic pathogens. With these organisms present in the environment, outbreak of disease can occur with acute stress to the host, or sudden instability in the housing system. The resulting clinical or subclinical disease to the host can have adverse effects on facility performance standards, and the integrity/repeatability of research results. Some zebrafish pathogens also have risk of zoonotic pathogen transmission to facility staff.  Disease risk can be minimized by developing a comprehensive Health Management and Biosecurity Program (HMBP) that considers all pathways of infection (water, fish, equipment, people, fomites, vectors, and feed), and routinely reports on fish health status for any system, facility, or establishment. A HMBP should utilize disease surveillance and screening of discrete populations based on statistical sampling models to catch potential lesions in biosecurity protocols, and provide evidence as the foundation for biosecurity decisions. This lecture will outline important considerations needed to develop and implement a Health Management and Biosecurity Program for the aquatic facility.



The Development and Maintenance of a Specific Pathogen Free Zebrafish Facility
Speaker: Carrie Barton, Zebrafish Facility Manager – Sinnhuber Aquatic Research Laboratory

Specific pathogen free (SPF) facilities are common for mammalian laboratory models, but this concept is still relatively new for zebrafish. In 2006, construction of a new zebrafish facility at Oregon State University began with the goal to create a facility that would prevent contamination by Pseudoloma neurophilia, a commonly found pathogen in research zebrafish colonies. To create this SPF program, new approaches for quarantine design and diagnostic testing methods were implemented. The new design centered around a three-stage quarantine system and multi-generation testing, while also eliminated the potential for contamination from quarantine to the main “clean” housing spaces. The SARL operates as a closed core facility, with strict biosecurity and internal health monitoring protocols in place to maintain the SPF status. One important outcome of this effort is that the SARL is positioned to be a resource for the research community by providing a highly robust colony of SPF to meet an ever-increasing research need.

© 2016 Charles River Laboratories International, Inc.