ILAR J
Volume 49, Number 3, 2008
Detection and Management of Microbial Contamination in Laboratory Rodents

Barthold.  Introduction: Microbes and the Evolution of Scientific Fancy Mice, pp. 265-271

Domain: 1 & 4
Species: Mouse – Primary

Historical Background:
1	Today’s inbred mice, “scientific fancy mice, have been significantly affected by microbial agents from their beginning over 100 years ago. Their foundations are built on thousands of years of purposeful breeding and trading of “fancy mice”. The dominant character in this movement was the Japanese waltzing mouse.
·	C.C. Little: Initially at Harvard Bussey Institute 1907 to 1914, grad student of William Castle, Castle researching “experimental evolution” in mammals, coat color inheritance with dogs, cats, guinea pigs, rabbits and mice. Little assigned to mouse breeding and performed seminal studies on the genetic basis of coat color. Developed 1st inbred lab mouse (dilute brown nonagouti aka dba aka DBA).  Went to work with Tyzzer and began “Mendelizing” the cancer problem, crossing dilute brown mice with Tyzzer’s tumor susceptible Japanese waltzing mice. Then took his dilute brown mice to Carnegie institute of Washington @ Cold Spring Harbor, NY.  Eventually established Roscoe B. Jackson Memorial Laboratory with Loenell Strong. 
·	Ernest E. Tyzzer: Researched genetic susceptibility to transplantable carcinomas and sarcomas among “races” of Japanese waltzing mice where a significant degree of inbreeding was already noted. Described cryptosporidium in stomach and intestines of mice for first time and a new disease in Japanese waltzing mice caused by spore-forming intracellular bacteria—Bacillus piliformis (now Clostridium piliformis)—Tyzzer’s Disease now documented in rats, gerbils, hamsters, GPs, and rabbits. Agent transmitted from soiled bedding.
·	Leo Loeb: Worked with Tyzzer on Japanese Waltzing mice. 
·	Loenell Strong: Worked with Little at Cold Springs, NY.  After arrival, epizootic of “mouse paratyphoid” (may have been Tyzzer’s disease, since Salmonellosis is more insidious in mice) decimated Little’s mouse colony (& Strong’s research). Strong noted various lines were fragile due to inbreeding and advocated strict sanitation standards. Brought the mice back from oblivion and continued the inbreeding and created tumor susceptible C3H strain, the long lived CBA strain and A strain mice. Due to limited dba mice, Strong embellished the gene pool for many mouse lines with wild mice captured from a pigeon coop at Cold Spring (thus agouti coloration of C3H derived from wild mice crossed with albinos and DBAs).   Strong moved to Stephen’s (now Bard) College in NY and brought his mouse lines and these were nearly destroyed by a fire in his mouse house (chicken coop retrofitted with sheet metal to keep rats out). DBA line was again nearly lost. Strong rebuilt lines and moved to University of Michigan with C.C. Little then Little and Strong moved to Bar Harbor and established Roscoe B. Jackson Memorial Laboratory benefitted from Strong’s collection of mice and lines developed by Little, including C57 (derived from Abbie Lathrop‘s mouse stocks). 
·	C.C. Little credited with creating the first inbred mouse strain (DBA) - BUT if not for Tyzzer and Strong DBA mice would not exist today and infectious disease would have robbed Little of his fame. Many mouse lines have no doubt been lost along the way due to infectious disease susceptibility.
·	Abbie Lathrop: Retired school teacher from MA failed at raising poultry and turned to mouse breeding for “fancy” rodent trade. Began with a few Japanese waltzing mice and expanded to avg. daily census of 11,000 mice at height of career. Acquired new genetic lines from other fanciers and added some wild stock, had inbred lines up to 12th filial generation, some of her mouse lines had propensity for cancer and provided these to cancer researchers (Loeb, Strong, EC MacDowell). Performed necropsies and kept detailed notes on infectious disease (tapeworms and diarrhea, hepatic abscesses).
·	John J Bittner: Strong’s grad student.  Demonstrated an infectious “milk influence” (mammary tumor virus) responsible for early onset mammary cancer.
·	Jacob Furth: Pathologist at Columbia, first to realize that infectious agents interfere with research results in genetically inbred mice. High leukemia Ak (later AKR) mice delays tumor onset when they developed pneumonia (stress-related lymphocytic apoptosis in thymus, target organ of AKR retrovirus). 
·	D. Kutscher: Documented 1st report of a bacterial disease in mice “pseudotuberculosis” (Corynebacterium kutscheri).
·	Erich Traub: At Princeton published studies on natural infectious of mice with lymphocytic choriomeningitis virus.
·	Rodent infectious disease knowledge contributed to by Wallace Rowe, Janet Hartley, Sara Stewart, Bernice Eddy, Ludwig Gross, John B. Nelson, Lisbeth Kraft and others through 1950s and 1960s.
·	Russell W. Schaedler: In 1960s created a “cocktail” of selected enteric bacteria from normal mice.  NCI modified the Schaedler flora (“altered Schaedler flora) to standardize the microbial status of mice at all NCI contractors.  
	
General:
·	Remarkable was the uniform susceptibility of the entire inbred DBA population—classic example of genetic susceptibility unique to inbred mouse populations.  
·	Considering mouse husbandry standards (barns, coops) microbial pathogens were no doubt prevalent and had ample opportunity for cross contamination among mouse stocks through trading of “scientific fancy mice” and purposeful or unintended introductions of feral and wild mice.
·	Most clinically obvious agents were first recognized- Cryptosporidium spp. Tyzzer 1912. Chronic resp. dz. (now due to Mycoplasma pulmonis, cilia-associated respiratory bacillus and other factors) recognized as significant dz. in rats as early as 1915 and later in mice (1937)
·	Researchers became aware of viruses in 1930 when infectious ectromelia caused amputation of extremities (dry gangrene) in surviving mice. Mouse encephalomyelitis virus with “spontaneous” paralysis recognized in 1934
·	General awareness of epizootic diarrhea of infant mice (EDIM) was there, but rotavirus etiology was not known. During EDIM studies, experimental mice developed posterior paresis leading to the discovery in 1949 of JHM virus, the harbinger of a very extensive MHV group of viruses.
·	1950’s & 1960s, expansive period of rodent history, discovery of adventitious viruses: lactate dehydrogenase-elevating virus, cytomegalovirus, mouse adenovirus, minute virus of mice, polyomavirus and reovirus---these became apparent in studies with cell cultures, transplantable tumors and cancer research.
·	Advances in rodent infectious disease prompted CDC and National Cancer Institute to hold symposium in 1965 of Viruses of Lab Rodents.
·	Only reference around this time (1960s) was the classic but out of print book Pathology of Laboratory Rodents by Cotchin and Roe.
·	Landmark conferenceà Viral and Mycoplasmal Infections of Laboratory rodents: Effects on Biomedical Research, held at NIH in Bethesda in 1984
·	In 1991 ILAR report on Infectious Diseases of Mice and Rats addressed the issue of infection versus disease, principles of rodent disease and prevention and effects of individual agents on research.
·	List of known infectious agents, overt and opportunistic pathogens, has grown and continues to grow. Laboratory mouse hosts well over 60 different pathogens.  Large list of agents documented in wild mouse populations. Growth due to recognition of opportunistic pathogens in genetically altered mice (Helicobacter) and advances in detection assays. Molecular methods continue to expand diagnostic horizons
·	Mouse poses unique challenges because of nature of the beast. Many inbred strains with intrinsic and extrinsic genetic susceptibility to various agents, mutant strains and immunodeficient strains and used for sophisticated research subject to relatively subtle experimental variables compared to other species. Drawing the line between overt pathogens, opportunistic pathogens and normal microflora/fauna of the mouse can be daunting, if not impossible.
·	The laboratory mouse is a mosaic of Mus musculus subspecies (4 subspecies). The lab mouse mosaic genome is a consequence of long history of global trading among mouse fanciers, journeys of commensal mice in concert with human global migration and colonization and introductions of feral mice along the way. There was the opportunity to acquire not only a staggering array of infectious agents indigenous to their diverse origins but also several new agents during domestication and trade among fanciers and scientists. Ectromelia virus appeared in lab mice in England in 1930 and now is worldwide. Periodic seasonal and multifocal outbreaks of Sendai virus in 1970s may have been due to human exposure, recent studies suggest that it may indeed be of human origin. Rotavirus infection of mice (EDIM) has unexplained seasonality. Exchange of pathogens continues to plague mouse populations through cross contamination during shipping, unrestricted traffic among institutions and investigators, exposure to untested biologic products and introductions and reintroductions through uninformed investigators.
·	Profound influence of retroelements: Evolutionarily there are recent replicating ecotropic retroviruses to ancient retrotransposons in genome of lab mouse. Retroelements make up over 37% of total lab mouse genome and contribute to mouse strain predispositions to various types of cancer.  Retroviral integrations are responsible for common mouse strain phenotypes (rodless retine- rd1, recessive allele) in Strong’s C3H mice, hairless mutation (hr) and dilute (d) coloration of Little’s DBA mice. Retroelement integrations contribute to 15% of spontaneous mutant phenotypes in lab mice. Take away retroviruses and there is little left to call a mouse.
·	Rodent infectious disease extensively controlled in animals from commercial vendors in 1970s and 1980s.  Rodents became remarkably pathogen-free thanks to vendor competition.  “Lists” of specified (known) pathogens were relatively short in “gnotobiotic” rodents. Population dynamics of mouse enteric flora are significantly influenced by mouse genetic and environmental differences.  Intestinal microflora can be standardized going in, but not going out.
·	With lab mice no clear line of demarcation between good and bad when it comes to infectious agents. 
·	The challenge is even greater with bacteria. Lots of money and effort expended on Pseudomonas—this ubiquitous environmental bacterium is pathogenic under very limited conditions (neutropenia induced by x-irradiation or cyclophosphamide). Pasteurella pneumotropica is an opportunistic pathogen that is normal intestinal microflora in wild and many conventional mice, but when eliminated its enteric niche is filled by Klebsiella, Proteus and other enterobacteria. Proteus mirabilus can cause fatal disease in immunodeficient mice.
·	Should we test for fungal agents? Far more overt pathogens in immunodeficient and genetically modified mice.
·	Mouse parasites- We test for Tritrichomonas spp. and Entamoeba muris, but there is no evidence that either cause disease or adverse effects on research. Leptospira ballum, significant zoonotic agent, although very common in feral mice, in not considered in lab animal facilities even with ample opportunity for infection.
·	Lab animal science is having difficulty defining membership on the “list”. Harmonization is needed with assessment of what agents are relevant or significant based on scientific information.
·	Are lab animal programs too restrictive for science and do they unnecessarily insist on unrealistically maintaining pathogen free rodents in agent-exclusive barriers? Use of barrier conditions or rederived rodents may actually modify phenotypes and research results achieved under conventional conditions.  Mice respond differently after rederivation which results in a state of immunological hyporeponsiveness.  Mice can be “too” clean creating adverse effects on research.  Rederivation results in the phenomenon of “the disappearing phenotype” with genetically engineered mice with “inflammatory bowel disease” due to natural Helicobacter spp. infection.
·	Most infectious diseases of lab rodents tend to be subclinical. There is naiveté or ambivalence of scientific community, so good surveillance and diagnostic programs are likely unappreciated.  Funding gets tight and costs rise, pressure to cut costs and eliminate quality control programs happen. 
·	Lab animal professional community role is declaring what is good and bad for mouse research, need to be defenders of good science. Lab animal vets often end up as policemen with very weak scientific justification to defend their position.	
·	The problem is not the concept but the mouse itself. The inherent value of the mouse is its “homogeneity of genetic constitution”. Ignoring microbial variables essentially negates the value of the model. Inbred and mutant status of accentuates these effects. There is no such thing as “normal microflora” for the lab mouse because of its long and circuitous history of domestication. Lab mice are not only genetic mosaics but also microbial mosaics so there is no such thing as a wild type mouse. Even if purposely reintroduce microflora, The task of ensuring their continued and uniform presence would be daunting. Exclusion is the most practical solution, but this requires surveillance, diagnosis, control and management.
·	Advent of transgenesis- many mutants with value to specific areas of scientific inquiry, but little global commercial value—given rise to a new era of the scientific fancy mouse.
1	NIH-funded Knockout Mouse Project and NIEHS Environmental Genome Project and Europe, Canada, Japan and China have similar projects and initiatives. Quality control and traffic of scientific fancy mice will be challenging. International traffic of new mouse lines, germplasm and embryonic stem cells—NEED standardization and quality control of testing modalities among countries and institutions.
2	Rule of “buyer beware”. “Specific Pathogen Free” is notoriously nebulous and meaningless w/o specifying pathogens. Credibility of test results is vital and affected by a variety of factors (testing laboratory, test method, sample size, animal age and sentinel or indigenous animals. Interpret results in context. Programs rely simply on serology and fail to recognize the value of diagnostic pathology to detect infections before they have spread throughout a population and resulted in seroconversion or to discover new pathogens
2	Rodent infectious disease surveillance and control require and understanding of biology of each agent in contact. Surveillance is often underemphasized and inadequately addressed by lab animal programs. National shortage of qualified lab animal vets to fulfill this need.
3	Essential element of infectious disease management is education. Scientists pay the bills and are entitled to info about rodent infectious agents, how they may affect research results and why animals need to be quarantined, tested and managed for scientists own protection. Informed scientists become advocates, but forcing restrictions and limiting access to their animals garners resentment.
4	Single standard of infectious agent exclusion seldom works for academic institutions, traffic is difficult to control and animals may be maintained in conventional, barrier or modified barrier conditions. 
5	Infectious agents affect research in a variety of ways and a central element of research is reproducibility. Journals should compel authors to include “specifics” of the pathogen status of their research animals and genetic background.  
6	International traffic of mice, germplasm and ES cell lines is increasing. There is the challenge of dealing with diversity and varied quality of material that is created by and exchanged among “cottage industry” research labs. The NIH NCRR established network of regional Mutant Mouse Regional Resource Centers (MMRRC) to serve the international biomedical research community. These centers provide ancillary services such as cryopreservation, rescue of failing or aged lines through assisted breeding methods, speed congenics, genotyping, construct design, electroporation, microinjection, health screening, pathology, phenotyping, colony management and disease consultation. The centers also have links to similar resources at Jackson Lab, in Canada, Europe, Japan and Australia. 
7	Scientific environment is rapidly becoming global—need for science-based international harmonization of variety of lab animal health and welfare issues and transportation and exchange of material involving all lab animal species. ILAR is embarking on updating the Guide for the Care and Use of Laboratory Animals- challenged with task of dealing with infectious disease quality control in lab rodent facilities. Last version of Guide remains timely and appropriately summarizes how to deal with microbial agents in rodents:
·	Scientific objectives of a particular protocol, the consequences of infection within a specific strain of rodent, and the adverse effects that infectious agents might have on other protocols in a facility should determine the characteristics of rodent health-surveillance programs and strategies for keeping rodents free of specific pathogens.

QUESTIONS:
1.	Which of the following, developed by Clarence Cook Little, is considered the first inbred laboratory mouse?
a.	C57
b.	dba
c.	AKR
d.	C3H
2.	True or False: “Specific pathogen fee” is a nebulous phrase and meaningless without specifying the pathogens.
3.	The Mutant Mouse Regional Resource Centers:
e.	Were established by the NIH National Center for Research Resources
f.	Serve the international biomedical research community
g.	Act as a repository of genetically engineered mice, germplasm and ES cell lines
h.	Are regionally located at UC Davis, University of Missouri and University of North Carolina
i.	All of the above

ANSWERS:
1. 	b. dba
2. 	True
3. 	e. All the above

Carty.  Opportunistic Infections of Mice and Rats: Jacoby and Lindsey Revisited, pp. 272-276

Domain 1, Prevent and control spontaneous or unintended disease or condition
Domain 4 T3, Manage or provide indirect management/oversight of laboratory animal facilities
Species: Primary, rats and mice

Summary: Jacoby and Lindsey performed a comprehensive review of the state of opportunistic rodent infections in research institutions in 1998. This is a summary document of data gathered during the first quarter of 2006. Results are slightly complicated by the definition of a “barrier” facility and how that definition is applied at each individual facility. 

1)	Health Monitoring and Outbreak Identification
a.	Quarterly testing is most common.
b.	Young adult immunocompetent mice are used by 97% as sentinels
c.	One sentinel cage for each side of the rack is most common, however this ratio was arbitrarily determined
d.	Molecular diagnostic testing (i.e., PCR)is most common for health monitoring; other methods include (in descending order of frequency):
i.	Serology 
ii.	Parasitology
iii.	Gross and microscopic pathology
iv.	Bacteriology
v.	Environmental testing
e.	Few respondents screen animals from commercial vendors
f.	97% screen, quarantine, or rederive animals from noncommercial sources.
i.	Contact sentinels are primary method
g.	Biologicals are frequently screened using PCR or MAB or RAB production
i.	Contamination has included MPV, LDH, MHV
h.	Costs are incorporated into per diems most commonly
2)	Agent prevalence among mice during the preceding 24-36 months
i.	Mice (in order of frequency)
i.	Viral agents
1.	Mouse parvovirus
2.	Mouse hepatitis virus
3.	Minute virus of mice
4.	Epizootic diarrhea of infant mice
5.	Noro virus was not addressed
ii.	Bacterial agents
1.	Pasturella pneumotropica
2.	Helicobacter spp.
iii.	Parasites
1.	Aspiculuris tetraptera
2.	Syphacia obvelata
3.	Mycoptes spp.
4.	Myobia musculi
j.	Rats
i.	Viral agents
1.	Rat parvovirus
2.	Kilham rat virus
3.	Sialodacroadenitis
ii.	Bacterial agents
1.	Pasturella pneumotropica
2.	Helicobacter spp
3.	Staphylococcus
iii.	Parasites
1.	Aspiculuris tetraptera
2.	Syphacia spp.
3.	Mycoptes spp
4.	Giardia spp
3)	Outbreak management, most common strategies for 3 highly transmissible diseases
k.	MHV – test and cull
l.	Pinworms – test and cull
m.	EDIM – take no action
4)	Summary
n.	Certain infections/infestations were reported more frequently, probably due to:
i.	Greater numbers and density of rodents housed in research facilities
ii.	Improved methods of detecting infectious agents
iii.	Larger numbers of rodents coming to research facilities from institutions other than commercial vendors
o.	Future improvements should include:
i.	Better and more consistent screening of rodent biologicals
ii.	Improved routine health monitoring for agents that do not transfer well by soiled bedding (Sendai virus, pinworms, mites)
iii.	Environmental monitoring

QUESTIONS:
1. 	Which of the following pathogens are not easily transferred by soiled bedding?
a. 	Sendai virus
b. 	Pinworms
c. 	Mites
d. 	Mouse hepatitis virus
2. 	Which of the following bacterial agents are frequently found in rat colonies and not mice colonies?
a. 	Pasturella pneumotropica
b. 	Helicobacter spp
c. 	Staphylococcus
3. 	Which of the following is an uncommon contaminant in biologicals?
a. 	Mouse parvo virus
b.  	Lactate dehydrogenase-elevating virus
c. 	Mouse hepatitis virus
d. 	Epizootic diarrhea of infant mice.
4. 	Which of the following testing methods is the most underused?
a. 	Parasitology
b. 	Bacteriology
c. 	Environmental testing
d. 	Serology 
e. 	Gross and microscopic pathology

ANSWERS:
1. 	a, b, c.
2. 	c
3. 	d
4. 	c

Besselsen et al.  Lurking in the Shadows: Emerging Rodent Infectious Diseases, pp. 277-290

Domain 1: Management of Spontaneous and Experimentally Induced Diseases and Conditions 

Primary species  Mouse, rat, hamster

SUMMARY: This article discusses the identification, detection, and impact of 4 emerged infectious diseases of rodents (parvoviruses, Helicobacters, Murine Norovirus, and Rat Respiratory Virus), as well as 3 emerging syndromes (Corynebacterium bovis, clostridial enteropathies, and inclusion body nephritis.  In addition, there is a brief overview of control of emerging infectious agents.

Rodent parvoviruses were initially detected as cell culture contaminants and, except for hamster parvovirus (HaPV), do not cause clinical disease but do modulate the T cell dependent immune response.  The viruses grow poorly in cell culture and most diagnostic tests use recombinant antigen (either rNS1 or rVP2).  NS1 (nonstructural protein) is highly conserved among rodent parvoviruses and can be used as a “generic” indicator of infection.  VP2 is used for parvoviral speciation.  Target tissues for diagnostic sampling include mesenteric lymph node and spleen.  MPV (mouse parvovirus) is frequently only intermittently detected in enzootically infected colonies due to the narrow window of high titer of viral shedding from infected animals and the need for a high viral dose to cause infection in sentinels.  

Since the time when Helicobacter hepaticus was detected as the causative agent of chronic active hepatitis in infected mice, additional Helicobacter species have been detected in fecal, intestinal, and liver samples from rodents.  H. hepaticus causes a lymphoplasmacytic typhlocolitis and hepatic dysplasia and neoplasia.  H. hepaticus can progress to proliferative typhlocolitis and proctitis, dependent upon both bacterial and host factors.  Helicobacter infections have been associated with the formation of gallstones and development of mammary cancer

Murine Norovirus has been shown to cause encephalitis, cerebral vasculitis, meningitis, hepatitis, splenitis, and pneumonia in mice deficient in STAT or IFN pathways.  Most strains show persistent infection and MNV DNA can be detected in feces, mesenteric lymph nodes, jejunum, and ileum.  All MNV strains show serologic cross reactivity

A disease of 6-20 week old rats characterized by multifocal interstitial pneumonia, histiocytic alveolitis, type II pneumocyte hyperplasia, and pulmonary vasculitis has been hypothesized to be a viral etiology (Rat Respiratory Virus).  Most rats recover but may be more susceptible to death during anesthesia.  The virus has not yet been isolated from affected rats.

Corynebacterium bovis is the causative agent for “scaly skin disease” which is characterized by orthokeratotic hyperkeratosis and acanthosis of the dorsal and ventral trunk.  C. bovis infection can alter the immune response and increase the toxicity of chemotherapeutics.

Clostridium perfringens type A has been associated with paresis or peristalsis in lactating mice.  C. perfringens type B and D and toxigenic C. difficile have been associated with necrotizing enteritis in recently weaned mice.  These diseases are hypothesized to result from disruptions to the normal intestinal microflora.

The final “disease” discussed is the large eosinophilic intranuclear inclusion bodies and karyomegaly in the renal tubular epithelium associated with interstitial lymphoplasmacytic infiltrates.  It has yet to be determined if this is a degenerative change or a viral etiology (polyomavirus).

QUESTIONS:
1.	What murine parvovirus causes persistent infection in lymphoid organs?
2.	What are diagnostic methods for detecting Helicobacter in rodents?

ANSWERS:
1.	MPV-1
2.	PCR of 16S rRNA gene, culture, serology, histological exam of target tissues

Clifford and Watson.  Old Enemies, Still with Us after All These Years, pp. 291-302

SUMMARY: Agents still prevalent in research animal colonies are parvoviruses (rats & mice), mouse rotavirus (EDIM), Theiler's Mouse Encephalomyelitis Virus (TMEV), Mouse Hepatitis Virus (MHV) and pinworms. Risk factors which potentiate continued infections with these agents include ambivalence about the need for eradication and increasing shipping of rodents between collaborators at different institutions. 

Agent-specific biology also affects persistence; for instance, parvoviruses are shed for long periods of time by infected animals and remain infectious in the environment for extended periods of time. In modern housing systems, prevalence of agents may be so low as to evade detection by sampling or sentinel systems. Infection resulting in a very low rate of viral shedding may fail to infect even cage mates of the infected individual. In spite of its fragility in the environment, MHV may persist due to its capacity for explosive spread within a facility. Pinworm ova are not consistently shed by infected adult animals, making diagnosis difficult. 

Sources of infection may be imported or feral rodents, contamination of supplies, contamination of biological reagents or cell cultures, or introduction by personnel who have handled contaminated materials or pet rodents. Difficulty of eradication may tax the financial and political capital of the animal facility. The impact of some infections on research parameters varies with the type of research but may be subtle or non-existent. However, detected infections have a major impact on inter-institutional transfers of animals for the purposes of scientific collaboration. 

QUESTIONS:
1.  	Which of the following viruses is/are rapidly cleared by immunocompetent rodents?
a. 	Parvovirus (MPV)
b. 	Rotavirus (EDIM)
c. 	Theiler's Mouse Encephalomyelitis Virus (TMEV)
d. 	Mouse Hepatitis Virus (MHV)
2.	Which of the following pathogens is/are relatively resistant to environmental decontamination?
a.	Parvovirus (MPV)
b. 	Rotavirus (EDIM) 
c. 	Mouse Hepatitis Virus (MHV)
d.  	Pinworms
3.   Which of the following pinworm species has the longest prepatent period?
a. 	Syphacia muris
b. 	Syphacia obvelata
c.  	Aspicularis tetraptera
4.   The most dependable method for diagnosing pinworm infection is:
a. 	Fecal flotation
b. 	Perianal tape test
c. 	Examination of cecal/colonic contents
5. 	T/F  The clearance of rotavirus requires both B and T lymphocytes and SCID mice are known to shed the agent for prolonged periods of time. 
6.	T/F  Cell lines, serum and other biologically derived reagents are major risk factors for the introduction of rotavirus. 
7. 	T/F  Sendai virus and Mycoplasma pulmonis have essentially disappeared from laboratory rodents in North America. 
8. 	T/F   Syphacia obvelata infection induces a Th2 cytokine response with elevated interleukins and circulating IgG

ANSWERS:           
1.	B & D
2.	A & D
3.	C
4.	C
5.	T
6.	F
7.	T
8.	T

Watson.  New Building, Old Parasite: Mesostigmatid Mites – An Ever-Present Threat to Barrier Facilities, pp. 303-309

Tasks: Domain 1 K1 and K3, Domain 4 K8
Species: Rodents

Abstract: Mesostigmatid mites are blood-sucking parasitic mites found in wild rodent populations. Periodically they can also become a problem for laboratory rodent colonies, particularly when building construction or renovations disturb colonies of commensal (building) rodents that had been acting as hosts. Mesostigmatid mites infest both rats and mice and, unlike the more common rodent fur mites (Myobia, Myocoptes, and Radfordia sp.), can survive for long periods in the environment and travel considerable distances in search of new hosts. They easily penetrate barrier caging systems, including individually ventilated cages, thus circumventing the usual precautions to protect rodents from infection. The two mites reported in laboratory rodent colonies, Ornithonyssus bacoti and Laelaps echidnina, also bite humans and have the potential to transmit zoonotic diseases. Once the mites gain access to a colony, eradication requires elimination of commensal rodent reservoirs in addition to insecticide treatment of both the laboratory rodents and the environment. In view of the undesirability of insecticide use in the animal facility, it is advisable to investigate the effectiveness of preventive treatments, such as environmental application of insect growth regulators or silica-based products. This article summarizes available information on mesostigmatid mites and their laboratory incursions, and provides suggestions for diagnosis, treatment, and control based on the authors experience with several outbreaks at a large academic institution.

SUMMARY: Despite a decrease of a number of pathogens found in research facilities, some remain and are difficult to detect due to their low prevalence and inefficient transmission by dirty bedding exposure. The wild rodent population is still the reservoir for a number of pathogens. This article focuses on mesostigmatid mites responsible for sporadic outbreaks due to wild rodent exposure especially during construction/demolition or renovations. Mesostigmatid parasites: All are dorsoventrally flattened with 4 pairs of legs attached to the anterior of the body. Under laboratory conditions, the life cycle is completed in 6 to 8 days. For more detailed info on the mites, read article Ornithonyssus bacoti (0.5mm), Laelaps echidnina (0.75-1mm) /large ventral genital shield (#O. bacoti). Infestation: Ornithonyssus spp. can be brought into research facilities by feral mice, pigeons and chickens as well. The mesostigmatid mice can travel long distances and live away from their host, the micro-isolator or barrier cages don't prevent the mites from entering/exiting the cage which complicates detection and eradication. Zoonotic: Humans can be bitten by these mites which can transmit a number of diseases (O. bacoti: Rickettsia akari and sp., francisella pestis/tularensis, Coxsackie virus, Trypanosoma cruzi, Coxiella burnetti, hantavirus, Borrelia, Bartonella and intermediate host for rodent filarial worm Litosomoides carinii ; L. echidnina: Junin virus, Hepatozoon muris and L. sanguineus: R. akari, C. burnetti) or/and lead to dermatitis. Detection/eradication/prevention: First sign= human bites, then observation of tiny moving black dots on animal or in the cage. Sticky traps only useful after start of treatment to determine extent of outbreak. Recommendation is to treat the environment (spray deltamethrin 0.06%) for mites before eradication of pests (mice etc) and mites in cage with MITEARREST before cage change monthly for 3 treatments which should be synchronized with demolition/renovation/construction. Environmental treatment should start from periphery to the center and comprise all areas occupied by rodents or rodent equipment. Beware of human exposure to treatment when making treatment choice, pyrethrins appear safe and effective. Prevention can be achieved by use of physical methods=silica-based products and biological methods=insect growth regulators (depending on the category chosen, beware of possible research consequences).

QUESTIONS: 
1.	Are micro-isolator cages protective in case of mesostigmatid mite infestations? Y/N
2.	What are the 2 main mesostigmatid mites seen in research?
3.	What is the common origin for the mesostigmatid mites?
4.	How to treat against mesostigmatid mites and what should be used?
5.	Are mesostigmatid mites able to transmit diseases? Y/N
 
ANSWERS: 
1.	No 
2.	Ornithonyssus bacoti and Laelaps echidnina 
3.	Wild rodents w/ or w/o renovation/demolition/construction 
4.	Treat environment first before treating cages, treat from periphery to center and all areas where rodents are present or rodent equipment is present. Pyrethrin in cage (mite arrest) and environment (deltamethrin) and prevention with silica-based products or insect growth regulators. 
5.	Yes

Peterson.  From Bench to Cageside: Risk Assessment for Rodent Pathogen Contamination of Cells and Biologics, pp. 310-315

Domain 1: Management of Spontaneous and Experimentally Induced Diseases and Conditions 
Task 1. Prevent spontaneous or unintended disease or condition 
Primary species: mouse and rat
 
SUMMARY:  This article discusses the risks of rodent pathogen contamination of biologics that are commonly used in an animal research facility. The decision as to whether a particular biologic should or should not be tested for rodent pathogens depends on its history of use. Some biologics may have never been exposed to any rodents or rodent-derived samples and as such probably do not need to be tested for pathogens. Other biologics may have been inoculated into rodents and then subsequently re-harvested, or may have been serially passed through rodent cell lines; these biologic agents are at the highest risk of acquiring rodent pathogens. Unfortunately a detailed history of a biologic¹s handling is often not available. Most institutions have strict guidelines and quarantine requirements for rodent import, but the same principles are often not applied to biologics.

Examples of biologics commonly used in an animal facility include cell lines, antibodies, serum, cytokines, basement membrane matrices (e.g. Matrigel) and mouse gametes/embryos. Hybridoma cell lines are particularly at risk of contamination as they originate from mouse splenocytes and myeloma cells, they are often cultured on mouse feeder cells and are sometimes re-inoculated into mice to boost their antibody-producing potential. As tumor cell lines are often inoculated into immunocompromised mice, the lack of an innate immune system would allow any rodent pathogen contaminant to proliferate unchecked.

Luckily, the incidence of biologic contamination is not as common today as in the past due to 1) aggressive health monitoring of rodents, 2) testing and culling of cell lines and 3) the use of the quicker PCR assay instead of MAP testing. One of the most common contaminant of cell lines is Mycoplasma sp., although cells lines are highly unlikely to be contaminated with rodent mycoplasmas.

There have been many documented cases of biologic contamination. Some examples are: 1) LDHV contamination of a basement membrane matrix, 2) an ectromelia outbreak that was due to contaminated pooled imported mouse sera and 3) LDHV contamination of monoclonal antibodies.
 
QUESTIONS:
1.	What is the most common biologic brought into an animal facility?
2.	What two infectious agents remain the most common causes of endemic infections in laboratory colonies?
3.	Name two rodent mycoplasmas.
4.	What infectious organism has recently been shown to be capable of transmission through mouse gametes, ovarian tissue and embryos?
 
ANSWERS:
1.	Tumor cell lines
2.	MHV and MPV
3.	M. rodenticum, M. pulmonis
4.	MPV

Shek.  Role of Housing Modalities on Management and Surveillance Strategies for Adventitious Agents of Rodents, pp. 316-325

Task 1 - Prevent, Diagnose, Control, and Treat Disease 
Primary Species: Rodent 

SUMMARY:  Indigenous rodent viruses (often contaminants of tumor cell lines and virus stocks) and adventitious agents may confound research even though they may not cause disease in the rodent host.  In addition, some indigenous rodent viruses may be zoonotic.  Therefore, specific pathogen-free (SPF) rodents are needed.  Room and cage-level barrier systems have been developed to exclude microbial contaminants.  Strict adherence to bio-security practices in conjunction with routine health monitoring is paramount in order to verify and to maintain SPF status.  HEPA-filtered mass air cubicles and racks, isolators and filtered cages have proven to be effective in excluding and preventing spread of infectious agents.  Rodents housed in uncovered cages even in barrier rooms are contaminated with microbes from the environment and from people.  Examples of such microbes include Staphylococcus aureus, Pseudomonas aeruginosa, b-hemolytic streptococci and Pneumocystis carinii.  These adventitious agents may be particularly problematic in rodents that are inherently immunodeficient, such as athymic nude mice, SCID mice or mice that have been rendered immunodeficient via exposure to radiation or chemotherapy.  ELISA, IFA and PCR have greatly aided health monitoring.  False negative results may be more common for inappropriately sampled colonies in which the prevalence of infection is low and the colony is housed in microisolation cages.  Soiled bedding transfer of MHV and MPV are more efficient in ventilated microisolator caging compared to static microisolator caging.  Microbial PCR testing of environmental samples is an adjunct to but not a replacement for monitoring sentinel animals.

In commercial rodent production facilities when rodents are housed in uncovered cages, rodents are chosen from breeder and stock animals of varying ages for health monitoring.  In research settings, sentinel animals are used for health monitoring.  Outbred mice are optimum sentinels for immunocompetent colonies because outbred mice seroconvert in the absence of clinical disease.  Some immunocompetent inbred strains, such as C57BL/6 are not appropriate serology sentinels because they are resistant to infection with Mouse Parvo virus (MPV) and thus do not seroconvert.  Another inbred strain that is inappropriate as a sentinel is DBA/2 mice because they succumb to lethal Sendai infection before seroconverting.  Serology sentinels should be replaced on a regular basis because of age dependent resistance to infection (hence lack of seroconversion) to some agents including MPV and mouse rotavirus.  Because infections are more likely to be more pathogenic in immunodeficient strains, the diagnostic sensitivity of health monitoring may be enhanced by using immunodeficient sentinels for infectious agents whose diagnosis depends on the production of pathologic lesions or demonstration of the organism.  

Contact of the sentinel animal with the colony animal is the most efficient and reliable means of transmission of infection.  Therefore direct contact of sentinel animals with imported animals in quarantine is the recommended method of sentinel exposure for critical microbiologic assessment.  However, direct contact of sentinel mice with colony mice may be unacceptable or impractical for routine health monitoring.  Hence, exposure of sentinel animals to dirty bedding from colony animals is most often used for routine health monitoring. One limitation of this method is that sentinels housed in micro isolation cages and exposed to dirty bedding may not seroconvert even if an adventitious agent is present in the colony because the agent is not transmitted efficiently in dirty bedding.  Detection of adventitious agents spread by aerosol may be enhanced by housing sentinel animal s in microisolation caging with unfiltered exhaust air vented from the test colony. Finally, in colonies housed in microisolation cages, only a small percentage of animals may be infected with agents that are readily transmitted by dirty bedding, and the number of animals actively shedding the infectious agent for a particular time may be even lower.  As a result, sentinel animals housed in microisolation cages and exposed to dirty bedding may be exposed to such a diluted sample that they fail to seroconvert.  If the prevalence of an infectious agent is low, the number of animals sampled for health monitoring should be increased.  Similarly, the frequency of health monitoring for particular agents is driven and is directly proportion to the historical frequency of contamination, which is related to the housing type. For example, gnotobiotic or immunodeficient colonies maintained in isolators or microisolation cages are more commonly contaminated with adventitious bacteria.  Therefore microbiologic sampling is performed more frequently than serology and parasitology for animals housed in these conditions.  Conversely, viruses are the most common adventitious infection in commercial barrier rooms and therefore serology is performed more frequently than bacteriology or parasitology.

QUESTIONS:
1.	Which is the most efficient /reliable way to exclude adventitious rodent pathogens?
a.	Microisolator caging
b.	Flexible film and semirigid isolators
2.	Adventitious agents include
a.	Staphylococcus aureus
b.	b hemolytic streptococcus
c.	Pseudomonas aeruginosa
d.	Pneumocystis carinii
e.	All of the above
3.	Distinguish the following
a.	Gnotobiotic (completely defined microflora)
b.	Axenic  (germ free)
c.	Specific pathogen free (free of particular pathogens but have a complex  and undefined microflora)
4.	Advantages of microisolator  caging include?
5.	Disadvantages of microisolator caging include?
6.	T/F: Positive PCR results are indicative of infectious particles
7.	Appropriate sample size is determined by
a.	# of animals
b.	Prevalence of infectious agent
c.	None of the above
8.	If the prevalence of an infectious agent  in 10% in a population of 100, the appropriate sample size to examine in order to have a 95% confidence level of detecting the agent would be 
a.	7
b.	10
c.	29
d.	None of the above
9.	In a population of 100 if the prevalence of an infectious agent is 35% an adequate sample size to detect the agent (95% confidence level) would be
e.	7
f.	10
g.	35
h.	None of the above
10.	T/F  Negative test results indicate the agent is not present in the colony.
11.	When the prevalence of an agent is low, health monitoring should
i.	Use 2 detection methods
j.	Increase the number of samples examined
k.	All of the above
12.	Concerning the use of sentinel animals for health monitoring, the best method of detection is
l.	Direct contact of sentinel animals with test animals
m.	Exposure of sentinel animals to dirty bedding from test animals
n.	Exposure of sentinels to exhaust air from test cages
13.	False negative test results may result from 
o.	Dilution of  an infectious agent in dirty bedding  from multiple cages
p.	Use of inappropriate mouse strains as sentinels
q.	A and B
14.	Which of the following is easily  propagated in culture
r.	Pneumocystits carinii
s.	Helicobacters
t.	Parvoviruses
u.	Murine Norovirus
v.	None of the above
15.	T/F  Rodents produced in barrier rooms in uncovered cages are gnotobiotic
16.	Compared to rodents housed in uncovered cages, rodents housed in static microisolation cages are exposed to higher 
w.	Temperature
x.	Humidity
y.	CO2
z.	NH3
aa.	All of the above
17.	Room level barrier to infection includes
bb.	HEPA filtration of incoming air
cc.	Limited access to trained, gowned and gloved personnel/limited traffic
dd.	Disinfection of room equipment and supplies
ee.	All of the above
18.	Murine norovirus is pathogenic in mice lacking
ff.	Adaptive/specific immunity
gg.	Innate immunity
hh.	A and b
ii.	Athymic nude mice
jj.	SCID mice
19.	T/F: Effective microbiologic quality control may be achieved for specific pathogen free rodents using either routine health monitoring or stringent biosecurity.
20.	To obtain accurate health monitoring results, one must
kk.	Sample a sufficient number of animals
ll.	Sample appropriate animals
mm.	Sample with appropriate frequency
nn.	All of the above

ANSWERS:
1.	b
2.	e
3.	Gnotobiotic: completely defined microflora
	Axenic: germ free
	Specific pathogen free: free of particular pathogens but have a complex  and undefined microflora
4. 	1)	Versatility
2)	Effective cage level barrier to infection and spread of adventitious agents
5. 	Impedes spread of infectious agent such that prevalence is kept low, thus increasing risk of false negative test result and confounds corroboration of discordant lab results to verify infection
6.	F
7.	b
8.	c
9.	a
10.	F
11.	c
12.	a
13.	c
14.	e
15.	F
16.	e
17.	d
18.	b
19.	F
20.	d

Fontes.  Institutional Responsibilities in Contamination Control in Research Animals and Occupational Health and Safety for Animal Handlers, pp. 326-337

SUMMARY: 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 such a program correlate well with programs designed to control microbial contamination in laboratory animals. Facilities must be designed to maintain and contain research animals and the specialized equipment and controls needed to protect both handlers and animals. No individual should have access to an animal facility without medical clearance from the occupational health program. An effective occupational health program screens those with animal contact in order to identify individuals who may be particularly susceptible to animal allergens or the infectious disease under study, or who may present an elevated risk to the animals. This medical evaluation, which is part of the individual’s medical record, must be private and confidential. In general, immunization against tetanus should be provided every 10 years, and rabies vaccination is recommended for those who may handle wild-caught or other animals of unknown status used in research. Animal handlers must report to occupational health services after an injury or exposure. The occupational health provider will assess the need for post-exposure prophylaxis, instruct the injured handler on post incident care, and describe symptoms that will require additional attention. All exposures to animals and research-related materials should be addressed through immediate cleaning with antiseptic soup and/or immediate flushing of mucous membranes with potable water in an eye wash for 15 minutes. Handlers may be sensitized to allergens or other immune system stimulants. Repeated exposures to sensitizing agent may lead to more significant reactions and additional controls may be necessary to protect sensitized employees, including minimizing the level of involvement in high-risk procedures. Occupational health services will also evaluate physical injuries such as burns, sprains, and fractures to determine the employee’s schedule for returning to work in order to limit additional risks.

The 5 P’s of Risk Assessment (Pathogens, Procedures, Personnel, Protective equipment, Place) are explained in this article.
 
(1)	Pathogens: the red flags for discussion are agents that have been involved in laboratory-acquired infections, have an airborne route of transmission, have a low infectious dose, present a significant risk to the individual or other animals, have unique medical surveillance requirements or recommendations, may persist outside containment if released, and/or present decontamination challenges. 
(2) 	Procedures: the red flags for discussion are procedures that may involve the creation of aerosols or droplets, the use of sharps, or opportunities for bites or scratches. 
(3) 	Protective equipment: the red flags for discussion concerns animal procedures performed outside primary containment, as they could expose the handler to allergens or biohazards or expose the animal(s) to contaminants in the room. 
(4) Personnel: the red flags for discussion are animal handlers who have a poor safety or performance record or who may be at greater risk of infection if exposed (e.g. because they are immunocompromised or have contraindications for immunization). 
(5) Place (laboratory features): the red flags for discussion are experiments with Risk Group 3 agents, any other agents that have unique facility design requirements, and unusual research animals with special housing and/or handling requirements.**

Animal handler’s comprehension and knowledge determine the success of any containment program, so this review also includes a discussion of critical teaching points and the importance of evaluating personnel to verify their proficiency and competence in required protocols. Users must receive adequate training to ensure effective operation, and medical clearance for their proposed work assignments. Supervisors, managers, safety officers and others responsible for training animal handlers must verify retention and proficiency in the desired practices. 

The challenges in training and retraining are to identify the most important messages to communicate and to develop a program that involves trainee interaction and thus provides direct experience.

Fur further information about protection of handlers and laboratory animals, the author suggests two references: (1) Occupational Health and Safety in the Care and Use of Research Animals (NRC 1997) and (2) the Guide for the Care and Use of Laboratory Animals (NRC 1996).

TRUE or FALSE:
1.	In general, rabies vaccination is recommended for all persons handling animals.
2. 	Repeated exposures to sensitizing agents may help alleviate symptoms of allergies in the long run in sensitized individuals.
3.	The success of any containment program in controlling contamination of laboratory rodents or in protecting workers from exposure to potential biohazards usually relies on how ell the primary messages are disseminated and retained by employees.

ANSWERS:
1. 	F
2. 	F
3. 	T

Nicklas.  International Harmonization of Health Monitoring, pp. 338-346

Domain 1: Management of Spontaneous and Experimentally Induced Diseases and Conditions
 
SUMMARY: With the globalization of research and the increased shipping of laboratory animals (rodents) worldwide, strategic, comprehensive health monitoring has become more important.  Although rodent colonies are dramatically cleaner than they were in the 1970s when health monitoring focused on finding the cause of clinical disease, certain pathogens, especially ectoparasites, have become increasingly prevalent in the last decade.  This is largely attributable to the breeding and sharing of transgenic mice in the face of inadequate financial and intellectual resources devoted to health surveillance.  The author proposes several general guidelines for health monitoring programs including the fact that no less than 5-10% of total husbandry expenses should be devoted to health monitoring, and that health monitoring programs should be overseen by laboratory animal veterinarians with training in rodent health surveillance. 
 
Although the specific agents tested for in health surveillance programs are relatively uniform worldwide, and in spite of repeated calls for standardization of health monitoring procedures, international harmonization does not yet exist.  Arguments against international standardization include the idea that surveillance programs need to be customized to individual populations and that one size does not fit all.  Many also think that standardized health monitoring would not allow for the flexibility necessary for different husbandry techniques (caging) and different research areas.  Nevertheless many programs are poorly thought out and improperly implemented leading to repeated disease outbreaks. 
  
The author advocates for harmonization of health monitoring programs based on the Federation of European Laboratory Science Associations (FELASA) recommendations.  FELASA recommendations also exist for large laboratory animals including nonhuman primates.  A sample size of ten animals per microbiological unit is recommended and language is included that “additional investigations may be deemed necessary” to provide flexibility for the inclusion of emerging agents (e.g. MNV).  Although the author believes that FELASA recommendations are often misunderstood and misinterpreted, he feels that this document could be used to develop internationally standardized health monitoring guidelines that will “strengthen research results, save animal lives, and help contain costs.”`   
 
QUESTIONS:
1. 	Objections to international harmonization of rodent health monitoring include a loss of flexibility to customize health monitoring programs to which of the following?
a. 	Different husbandry techniques (e.g. caging)
b. 	Different research areas
c. 	Different rodent populations (e.g. immunocompromised mice) 
d. 	All of the above
e. 	None of the above
2. 	True or False?  Federation of European Laboratory Science Associations (FELASA) health monitoring recommendations exist for large animals, including nonhuman primates, in addition to laboratory rodents.
3. 	True or False?  There has been a decrease in ectoparasite infections in laboratory rodents in the last decade.
4. 	What percentage of the total husbandry budget does the author advocate spending on health surveillance?
a. 	At least 5-10%
b. 	Less than 3%
c. 	No more than 20%
d. 	At least 25%
d. 	None of the above
 
ANSWERS:
1. 	d 
2. 	True
3. 	False; rodent ectoparasite infestations are more prevalent than they were a decade ago
4. 	a

Mahabir et al.  Rodent and Germplasm Trafficking: Risks of Microbial Contamination in a High-Tech Biomedical World, pp. 347-355

SUMMARY: Preservation of mouse germplasm is achieved by cryopreservation of spermatozoa, embryos, or ovaries, and embryonic stem cells are used for the production of genetically engineered mice.

The most prevalent pathogens in the United States and Europe  are mouse hepatitis virus (MHV1), parvoviruses, mouse rotavirus, Theiler’s murine encephalomyelitis virus (TMEV1), reovirus type 3 (reo 3), Sendai virus, and mouse adenovirus (MAdV) (FL + K87). Lymphocytic choriomeningitis virus (LCMV), mouse K virus, ectromelia virus, MAdV, and  mouse thymic virus (MTV) have not been detected in Europe in the last decade.

Viral shedding: short - MHV, mouse rotavirus, and Sendai virus; moderate ectromelia virus and PVM;.  longer periods of time - LCMV, lactate dehydrogenase–elevating virus (LDHV), murine cytomegalovirus (MCMV), minute virus of mice (MVM1), mouse parvovirus (MPV1), TMEV, and MTV. 

Shipment of Live Mice
European guidelines - Appendix A of the European Convention for the Protection of Vertebrate Animals Used for Experimental and Other Scientific Purposes. Imports to countries of the European Union require the permission of the local government.UK Laboratory Animal Science Association (LASA) - Guidance on the Transport of Laboratory Animals.

US guidelines for the transportation of laboratory animals (NRC 2006). Other US regulatory agencies: Centers for Disease Control and Prevention (CDC), Department of Transportation (DOT), US Fish and Wildlife Service. Research mice are exempt from these regulations provided that they are not carrying infectious diseases and are from the genus Mus musculus. 

US customs clearance documentation: invoice stating that the shipment contains “Live Laboratory Mice” and listing the species (Mus musculus), number, gender, age, type of package, and names and addresses of the exporting and importing institutions. The invoice should include a statement that the animals do not meet the criteria for an endangered species nor pose a risk to human health. Health certificate and veterinarian’s statement that “Animals are healthy and have not been exposed to or inoculated with any livestock or poultry disease agents exotic to the United States” and that “The animals have not originated from a facility where work with exotic disease agents affecting livestock or poultry is conducted.”

Air Transport Association (IATA) regulations should be followed when packing and preparing live animals for air transport: 

o	Health report by a veterinarian of the exporting institution’s animal colony including a one and a half year history of the colony’s health status.
o	Packaging in adequate transport boxes, with bedding, food, and water.

Ground transport for regional shipping pose the least stress as it entails minimal handling of the shipping container and a single environment during transportation. Ground transportation should be carried out by licensed personnel in climate-controlled vehicles using the shortest routes. Long distance or transcontinental shipping generally requires coordination of both ground transport and air freight and may be performed by multiple subcontractors, resulting more handling. Mice that experience shifts in light/dark cycles require up to 2 weeks to normalize and thus require a longer period of overall adaptation and restoration, ranging from days to months, depending on the stress.

Shipment of Cryopreserved Materials
Cryopreserved germplasm (spermatozoa, oocytes, and resulting embryos) have advantage of preserve mouse models without the welfare concerns that arise in the shipping of live animals and for preliminary testing of sample aliquots to determine microbiological status before use.

Established repositories in countries around the world have formed the Federation of International Mouse Resources (FIMR) and can assist in the recovery of cryopreserved materials.

Cryopreserved oocytes, embryos, spermatozoa, and ovaries are shipped in a “dry shipper” in which the liquid nitrogen is absorbed in the shipper liner and maintained an ultralow temperature for 7 to 21 days. Documentation of an inversion test should be included to demonstrate that no free liquid nitrogen is present in the shipper. Cell lines, including murine embryonic stem (ES1) cells, and tissues are usually shipped on dry ice with special packaging and labeling requirements. embryos can be transported for less than 24 hours  in holding medium in cryopreservation straws at room temperature. Murine ES cells can be sent directly in the culture flask at room temperature.

Importation paperwork for cryopreserved laboratory mouse tissues and cell lines is similar to that required for live animal importation to the United States (i.e., a pro forma invoice and declaration statements).

Microbial Contamination of Germplasm
After removal of the surrounding cumulus cells, the zona pellucida is composed of a complex fibrous network interspersed with numerous pores that are largest at the outer surface and decrease in size centripetally.

In mouse germplasm cells, pathogens may be present in: (1) oocyte at fertilization and replicate in the embryo, fetus, or pups; (2) spermatozoa and carried into the oocyte at fertilization; (3) adhere to the zona pellucida; or (4) embryo after damage to the zona pellucida during handling

There are typically four main experimental designs to determine the risk of pathogen transmission by in vivo derived embryos:
• 	in vitro–in vitro: embryos from clean donors are exposed to the pathogen in vitro and assayed in vitro
• 	in vitro–in vivo: embryos from clean donors are exposed to the pathogen in vitro and transferred to seronegative recipients, which are screened for the development of antibodies to the pathogen
• 	in vivo–in vitro: embryos are collected from infected and/or seropositive donors and assayed in vitro
• 	in vivo–in vivo: embryos are collected from infected and/or seropositive donors and transferred to seronegative recipients, which are screened for the development of antibodies to the pathogen.

Efficacy of Washing Protocols and Embryo Transfer in Eliminating Viruses
Because of the zona pellucida porous nature and depending on the virus size, even after washing there may be a risk of viruses nesting in the ZP micropores. For example, reports show that trypsin treatment of embryos has not been successful in removing Sendai virus even after 12 washings.

Embryo transfer recipients in rederivation programs should be held in individually ventilated cages (IVCs1) until testing shows that they are free of all unwanted microorganisms. There should be a minimum of 6 weeks between the ET and microbiological examination of the recipients, coinciding with weaning of the offspring at 3 or 4 weeks of age (wild-type littermate pups should not undergo health monitoring until they are at least 6 weeks old).

QUESTIONS:
1.	How can a mouse germplasm be preserved? 
2.	Which are the most prevalent pathogens in USA and EU?
3.	Which pathogens have not been detected in USA and EU in the last decade?
4.	Give example of pathogens based on shedding period.
5.	Name four regulatory agencies responsible for transportation of live animals. 
6.	Give definition of germplast.

ANSWERS:
1.	Cryopreservation of spermatozoa, embryos, or ovaries, and embryonic stem cells. 
2.	MHV1, parvoviruses, mouse rotavirus, TMEV1, reo 3, Sendai virus, and MAdV, (FL + K87).
3.	LCMV, mouse K virus, ectromelia virus, MAdV, and  MTV have not been detected in Europe in the last decade.
4.	Short - MHV, mouse rotavirus, and Sendai virus; Moderate - ectromelia virus and PVM;.  Longer periods of time - LCMV, LDHV, MCMV,  MVM1, MPV1, TMEV, and MTV. 
5.	CDC, DOT, US Fish and Wildlife Service, IATA
6.	Spermatozoa, oocytes, and resulting embryos