PURPOSE: Contamination of biological specimens, such as cell lines, hybridomas and tumor cells, with rodent pathogens can result in devastating outbreaks of disease in laboratory animals implanted with these materials, as well as confounding and causing deleterious effects on tissue culture-based experiments. Research Biologics include: cells, tissues, stem cells, proteins, serological components of cell culture media, viruses (e.g. lentiviruses and adenoviruses), or other material that is derived from a living system. Biologics are commonly passaged through rodents for maintenance or are derived directly from an animal model. As a result, there is the potential for these biologics to contain animal pathogens thereby requiring measures to mitigate this risk.

I. Background

Due to the use of research biologics in animals, there is the potential for biologics to become contaminated with rodent pathogens capable of introducing disease into rodent colonies and human handlers. Although the prevalence has decreased in recent years, 25% of 297 mouse, rat, hamster, and human transplantable tumors as well as 69% of 465 murine leukemia’s and tumors have historically been found to be contaminated with mouse or rat pathogens in the past.⁵ Additionally, Corynebacterium bovis (C. bovis) was found to be a contaminant of 4% and 13% of cultured cell lines or solid tumor tissue when submitted for human or rodent pathogen testing.^1,3 An outbreak of C. bovis in Italy was thought to be due to cross-institutional collaboration suggesting that sharing tumor lines could be a potential route of contamination between susceptible mouse colonies.⁶

Rodent colonies within all animal facilities at McMaster University are routinely screened for infectious diseases and are maintained free of viruses and other microbial agents capable of interfering with research unless specified otherwise. Despite this, the health status of rodent colonies and the integrity of research can be endangered by inadvertent introduction of untested biological material carrying rodent pathogens. As such, the need for proper screening and handling of research biologics when used in animals is of upmost importance in maintaining biosecurity within the animal facilities and the integrity of research results.

II. Guidelines

Biological Materials that require testing:

•  All biological materials of unknown pathogen status used at McMaster Animal Facilities are to be tested for specified rodent pathogens as determined by the McMaster University Animal Facility (AF) Veterinary Staff prior to inoculation into rodents.
  • Unknown pathogen status is defined as any biological material that has no accompanying documentation proving it is free of rodent pathogens
  • PLEASE NOTE: The American Type Culture Collection (ATCC) DOES NOT test cell lines for the presence of rodent pathogens

Conditions that exempt Biological Materials from testing:

•  Biologicals may not need testing if:
  • There is credible documentation indicating they are free from rodent pathogens
    •  In order for documentation to be credible, it must include: testing date, list of screened pathogens, diagnostic test(s) performed, and the laboratory that performed the test.
    •  This documentation must be provided to and approved by the McMaster University AF Veterinary Staff
    • Each biological will need to be assessed on a case by case basis to determine if further testing is needed
  •  They are freshly prepared and have never been passaged through or established in rodents. Equipment (carts, ice buckets, lab coats etc.) used to prepare biological materials must have also been separate from equipment that comes into direct contact with rodents or rodent facilities.

Special Requirements

• Human Patient-Derived Xenograft Tumors and Human Derived Cell Lines
  •  All human patient-derived xenograft tumors and cell lines received from outside institutions must be C. bovis negative before implantation into rodent models
    •  Acceptable testing methods include qPCR assay for C. bovis DNA or bacterial culture
    •  Please Note: testing is based on submission of a piece of tumor tissue. qPCR negative results may not be representative of the entire sample

AUP Requirements

• All new Animal Utilization Protocol (AUP) submissions to the Animal Research Ethics Board (AREB) requesting the introduction of research biologics into rodents will be required to submit evidence of testing (copy of credible documentation as described) with the AUP submission or proof that the research biologic has not previously been passaged in rodents as previously described.  Protocols in which testing or proof has not been completed/provided will not be approved by AREB.
• All research groups currently using any form of research biologic in rodents will be required to submit evidence of testing (copy of credible documentation) as part of the Annual Protocol Renewal process.  Protocols in which testing has not been completed will not be renewed by AREB until evidence is provided.

III. Testing

Types

• Samples can be submitted to either Charles River Research Animal Diagnostic Service or IDEXX Bioanalytics (Formerly RADIL University of Missouri) for PCR testing. A “McMaster University” testing profile has been created at both locations (2012) which tests for the following rodent pathogens: Mycoplasma Genus Spp; Lactate dehydrogenase-elevating virus (LDV); Murine coronavirus; Murine Norovirus; Mouse Parvovirus (MPV1-3); Minute virus of mice (MVM); Mousepox (Ectromelia); Mouse Kidney Parvovirus (MuCPV); Mouse Hepatitis Virus (POLY); Pneumonia Virus of Mice; Sendai virus; Theiler’s murine encephalomyelitis virus/GDVII; C. bovis; M. pulmonis.
• Further information on testing can be found at:
  • https://www.criver.com/products-services/research-models-services/animal-health-surveillance/cell-lineresearch-biologics-screening?region=3601
  • https://www.idexxbioanalytics.com/biological-testing

Preparation of Samples

• If samples have been inoculated with any infectious agents which may pose a threat to human health, this information must be made known at the time of submission so that appropriate arrangements can be made in advance of shipping.
• Samples are to be submitted to the CAF veterinary staff in a screw top cryovial clearly labelled with cell line/antibody identification and cell concentration:
  • Cells:
    •  Two (2) undiluted 200uL samples.
    •  Cells may be in the form of a pellet or in growth media, freeze media or phosphate-buffered saline.
    •  A minimum of 1 x 10⁶ cells /mL should be submitted, please note on the submission form if there are more than 5 x 10⁷ cells /mL.
  • Antibody:
    • Two (2) undiluted 200uL samples; if these volumes are not possible, contact Charles River or IDEXX for recommendations
    • Please indicate on the submission form if the protein concentration is greater than 1.5 mg/mL.
  • Tissue/Solid Tumor Samples:
    • One (1) 2.0 mL snap top or screw top tube of each sample with a minimum of 30 mg of tissue (2-3mm size fragment or larger).
    • Conditions: Samples need be collected aseptically to prevent inadvertent contamination. If possible, samples should be passaged without antibiotics prior to shipping.
    •  If possible, samples for Mycoplasma testing should be passaged without antibiotics prior to shipping. Cells do not need to be viable for PCR testing. If shipping samples containing DMSO, please indicate this.
    • Samples should be stored at -20º Celsius, packed on dry ice and brought to the CAF on a designated day for shipment to CRL and IDEXX.
  •    Coordinating submission of samples to CRL/IDEXX by the CAF
    •  Please contact the Technical Manager McMaster CAF; DBRI (905-525-9140 ext. 22564) for further information

References:

1. Debrue MC, Henderson KS, Lipman NS, Manuel CA, Mulder GB. 2015 Corynebacterium bovis: is scaly skin still keeping you down? Panel discussions p 47. Presented at the 66th Annual AALAS National Meeting Program, Phoenix, Arizona, 1–5 November 2015. In: 66th Annual AALAS National Meeting Program, Memphis (TN): American Association for Laboratory Animal Science.
2. Dole VS, Henderson KS, Fister RD, Pietrowski MT, Maldonado G, Clifford CB. 2013. Pathogenicity and genetic variation of 3 strains of Corynebacterium bovis in immunodeficient mice. J Am Assoc Lab Anim Sci 52:458–466.
3. IDEXX BioResearch Resources. [Internet]. 2017. PDX and tumor stocks: biosecurity risks webinar series. [Cited 04 February 2017]. Available at: https://www.idexxbioresearch.eu/resourcesbiological-testing/
4.  Pritchett-Corning KR, Cosentino J, Clifford CB. 2009. Contemporary prevalence of infectious agents in laboratory mice and rats. Lab Anim 43:165–173.
5.  Niklas, W., V. Kraft, and B. Meyer. 1993. Contamination of transplantable tumors, cell lines, and monoclonal antibodies with rodent viruses. Lab Anim 43(4):296?300
6. Scanziani E, Gobbi A, Crippa L, Giusti AM, Giavazzi R, Cavalletti E, Luini M. 1997. Outbreaks of hyperkeratotic dermatitis of athymic nude mice in northern Italy. Lab Anim 31:206–211.