Comparative Medicine
Volume 57, Number 1, February 2007
OVERVIEWS
Carroll et al. Mammalian Model Hosts of Cryptococcal Infection, pp. 9-17
Summary: In the thousands of fungal species that have been identified, fewer than 20
are considered pathogenic for humans. Mycoses are of concern in individuals with
impaired immunity, with the relatively recent emergence and re-emergence of disease
caused by Coccidoides immitis, Aspergillus fumigatus, Candida albicans, and
Cryptococcus neoformans. C. neoformans is a yeast pathogen in the phylum
Basidiomycota and has distinct mating types that produce fertile spores under specific
conditions. It was separated into 2 varieties with distinct capsular serotypes- var.
neoformans (serotypes A and D) and var. gattii (serotypes B and C). More recent work
has added the C. neoformans serotype AD. C. neoformans is a free-living organism
believed to come from soil contaminated with avian excreta, or from decaying wood and
plant material. Cryptococcosis was rare before the onset of human immunodeficiency
virus, and there has been a decline in prevalence in North America in the 1990’s as a
result of antiretroviral therapy available there. Without that antiviral therapy, it remains a
leading cause of community acquired meningitis.
The pathogenesis of C. neoformans is mediated by 4 main virulence factors: 1) the
ability to grow at 37°C, synthesis of an extracellular capsule, production of melanin, and
secretion of degradative enzymes. The capsular structure helps C. neoformans resist
opsonization and phagocytosis, and shedding of it during infection may subvert the
immune response. Melanin may help resistance to host oxidative stress from phagocytic
cells. C. neoformans disseminates hematogenously from its entry into the lungs to the
brain, skin, kidney, and liver. Protective immune responses that are typically successful
in fighting off infection in an immunocompetent host require a T helper (Th) 1 pattern of
cytokine- and lymphocyte-mediated adaptive immune response. Despite antifungal
treatment, immunosuppressed patients that survive infection may develop a chronic or
relapsing form of meningoencephalitis.
C. neoformans is also a veterinary pathogen that infects cats, dogs, cows, horses, and
primates, among other species, with a clinical manifestation that resembles human
illness. This has led to the use of animal models in studying the disease. The most
convenient method for experimental infection is intraperitoneal injection, yet it is the least
clinically relevant method. Meningoencephalitis infections usually involve intracerebral or
intracisternal injections, and pneumonia is modeled through intranasal or intratracheal
inoculations. It is classified as a Biosafety Level 2 pathogen and the use of a class II
biosafety cabinet for the manipulation of C. neoformans is recommended.
Immunocompromised people should avoid rooms housing mice with pulmonary C.
neoformans. Human meningitis infections in immunosuppressed people have a mortality
rate of 10-30% despite antifungal treatment. Current initial therapy recommendations are
to use amphotercin B in combination with flucytosine for 2 weeks followed by
fluconazole. Anitfungal therapy studies for meningoencephalitis infections have used
rabbit and mouse models.
This paper describes 4 common mammalian hosts used in experimental cryptococcal
infection. 1) Guinea pigs were the first animal model system used in studies of
cryptococcosis. Female guinea pigs are more resistant to intraperitoneal infection with C.
neoformans than are males, which is consistent with a gender effect in human disease
susceptibility. 2) Rabbits are naturally resistant to cryptococcosis, which may be due to
their high normal body temperature (39.3-39.5 °C) inhibiting fungal replication. Immune
suppression through corticosteroid administration is commonly used to circumvent this
natural resistance. A rare condition of cryptococcal endophthalmitis was modeled in
healthy outbred New Zealand White female rabbits through intracarotid injection. Rabbits
can be repeatedly sampled for CSF in meningitis studies. 3) Inbred mice are the most
popular model for cryptococcal studies and inbred strains are highly susceptible to
infection through the intratracheal, intranasal, intravenous, and intraperitoneal route
without immune suppression. Mouse neutrophils and alveolar macrophages have the
ability to phagocytose and kill C. neoformans, and resistance to progressive lung
infection in mice is associated with the development of a Th 1 pattern of adaptive
immunity. A robust cell-mediated immunity interacting with humoral host defenses is
critical for protection from and clearance of cryptococcal infection. 4) Rats are used for
studying chronic or latent pulmonary cryptococcal infection with potential for reactivation
after corticosteroid treatment. Immunocompetent rats don’t predictably disseminate
pulmonary infection after intratracheal administration of the organism, unlike inbred
mouse strains, so rats may be more resistant to progressive cryptococcal disease.
Unlike rabbits, but similar to guinea pigs and mice, rats don’t require immunosuppressive
treatment to be susceptible to C. neoformans. Both rats and mice have been useful in
analyzing the immune response during respiratory tract and disseminated disease.
Progress in microbial genomic in combination with mammalian host models should
advance understanding of the disease.
Questions:
1) T/F Cryptococcus is a free-living yeast.
2) The virulence factor(s) of C. neoformans is/are:
a) capsule
b) melanin
c) 37°C growth
d) enzymes
e) all of the above
3) Cryptococcus is a ------------ pathogen.
a) BSL 1
b) BSL 2
c) BSL 3
d) BSL 4
4) Four common mammalian models in C. neoformans studies are:
a) Rats, mice, rabbits, and hamsters
b) Rabbits, dogs, cats, and guinea pigs
c) Rabbits, rats, mice, and guinea pigs
d) Primates, mice, rats, and rabbits
5) T/F Rabbits are naturally susceptible to C. neoformans infections.
Answers:
1) T
2) e
3) b
4) c
5) F
He et al. Pathobiology and Management of Laboratory Rodents Administered
CDC Category A Agents, pp. 18-32
CDC Category A agents include 3 bacteria, 1 bacterial toxin, and 3 types of viruses.
These agents include: Bacillus anthracis (anthrax), Clostridium botulinum toxin
(botulism), Yersinia pestis (plague), variola major virus (smallpox), Francisella tularensis
(tularemia), and the filoviruses and arenaviruses. All CDC Category A agents are also
agents that could pose a threat to public and animal and plant health and safety
(http://www.cdc.gov/od/sap/). Agents are reviewed with a focus on comparing the
biologic effects in naturally infected humans and rodent models and on considerations
specific to the management of infected rodent subjects. The laboratory animal species
used most frequently include the mouse (Mus musculus), rat (Rattus norvegicus), guinea
pig (Cavia procellus), and hamster (Mesocricetus spp.). Knowledge of the correct
animal pathobiology due to infectious organisms facilitates performance of a complete
and accurate risk assessment and the development of appropriate animal management
techniques.
Questions
1. Which of these agents does not below to Category A?
a. Rabies virus
b. Bacillus anthracis
c. Clostridium botulinum
d. Arenaviruses
2. Which the these pieces of equipment is not suitable equipment recommended for
containment of infectious agents?
a. Specialized inhalation exposure systems
b. Laminar flow hood for SPF animals
c. Fit-tested respirators
d. Biologic safety cabinets
3. Select the option which correctly completes the following statement: Personnel who
work with Category A agents…
a. Should receive a pay differential
b. Are in an extreme risk situation
c. Must receive a hazard pay
d. Work under BSL1
4. Select an inconvenient use of guinea pigs as a model for human infection with Ebola
virus.
a. Does not exhibit the hemorrhagic syndrome
b. Does not allow assessment of disease progression
c. Does not allow assessment of viral infection
d. Does not exhibit antibody production
5. Brown Norway rat is the best model for human infection of?
a. Francisella tularensis
b. Ebola virus
c. Yersinia pestis
d. Variola major virus
6. F344 rat is the best model for which human infection?
a.
b.
c.
d.
Clostridium botulinum
Ebola virus
Yersinia pestis
Francisella tularensis
Answers: 1. a; 2. b; 3. a, 4. d
Answers were not given for questions 5 and 6
Stump and VandeWoude. Animal Models for HIV AIDS: A Comparative Review,
pp. 33-43
Task 9: Collaborate on the Selection and Development of Animal Models
K1 - Animal Models (spontaneous and induced)
Summary: Early & dramatic CD4+ T cell loss at mucosal sites and failure of these cell
populations to recover play a key role in setting the stage for HIV disease progression.
The high rate of viral replication, high error rate of reverse transcriptase, and host
immune pressure contribute to rapid viral evolution and mutation, leading to alteration of
key viral proteins important for targeting by the host immune system. The HIV envelope
protein (Env), the main target of neutralizing antibody, is highly glycosylated, further
restricting effective neutralizing antibody binding. Different animal models have been
used to model HIV. Inoculation of adult C57BL/10 or C57BL/6 mice with Murine
Leukemia Virus (MuLV) resulted in lymphoproliferative disease and profound
immunosuppression, which is also referred to as “Murine AIDS” (MAIDS). Although this
model provides opportunity to study the contribution of both viral and host genetics to
retrovirally induced immunodeficiency disease pathogenesis but marked differences in
viral structure and pathogenesis limit its overall utility as an animal model of AIDS. FeLV
is an important model for studying mechanisms of latent versus progressive infection but
the drawback is the difference in viral genotype with respect to lentiviruses. SRV type D
is no longer used as an animal model of AIDS. Although all lentiviruses infect monocytes
and macrophages, HIV, SIV, and FIV also infect T cells. The primarily monocytemacrophage-tropic lentiviruses- Equine Infectious Anemia (EIAV), Maedi-Visna Virus
(MVV), Caprine Arthritis-Encephalitis Virus (CAEV), Bovine Immunodeficiency Virus
(BIV), and Jembrana Disease Virus (JDV) are associated with diseases resulting from
chronic inflammation. In contrast, T-cell tropic viruses (HIV, SIV, and FIV) cause disease
characterized by immune deficiency with secondary infections and tumors. EIAV is the
only known vector-borne (Tabanid flies) lentivirus and causes acute progressive disease
with unique feature of viral recrudescence. The MVV and CAEV models provide the
opportunity to study genetic susceptibility and host adaptation because clear breed
susceptibility has been established. FIV uses CXCR4 as an entry receptor and
CD134/OX40L as a binding receptor. As these molecules are on CD4+ T cells, FIV- like
HIV =96=primarily targets CD4 cells. The virus binding and entry mechanisms are
similar to HIV. FIV experimental model has been used extensively for studies of
pathogenesis, immunopathology, prophylaxis, and vaccine development. SIV in
macaques (SIVmac) resulted from cross-species transmission of the SIV strain
associated with naturally occurring SIV infection of healthy sooty mangabey monkeys
(SIVsmm). Genomic sequencing of SIVsmm and SIVmac revealed close relationship to
HIV2, establishing SIVsmm as the origin of HIV2 in humans. SIV of chimpanzees
(SIVcpz) has been established as the cross-species origin of HIV1 in humans. SIVs
predominantly use a CD4/CCR5 receptor entry mechanism. SIVmac infection of rhesus
macaque is considered the most important primate model of HIV disease. This model
has been used to evaluate vaccine strategies and to understand lentiviral-induced CNS
disease & maternal transmission. Naturally occurring SIV in African monkeys has been
studied for clues to host-virus adaptation mechanisms. Efforts to establish HIV1 infection
in primates have been unsuccessful. SIV-HIV chimeras (SHIV) have been developed
that express the HIV1 envelope glycoprotein and accessory genes within the backbone
of SIV. SCID-human chimeric mice provide the opportunity to experimentally infect
human tissues with HIV.
Questions:
1. In retroviruses, reverse transcriptase is encoded as part of
a) Gag gene
b) Pro gene
c) Pol gene
d) Env gene
2. Which genus of Retroviridae family causes cytopathic effects in vitro and are
clinically characterized by slowly progressive disease.
a) Alpha retrovirus
b) Gamma retrovirus
c) Epsilon retrovirus
d) Lentivrus
e) Spumavirus
3. In HIV disease, the main target of neutralizing antibody is the envelope protein (Env)
which is highly glycosylated to restrict effective neutralizing antibody binding. T/F
4. _________ infected with SIV or SHIV have been used most widely for development
of vaccines currently in clinical trials.
a) Chimpanzees
b) Rhesus macaques
c) Pig-tailed macaques
d) Baboons
5. Murine AIDS caused by MuLV is a very good model to study AIDS due to marked
similarities in viral structure and pathogenesis. T/F
6. FeLV is an important model for studying mechanisms of latent versus progressive
infection. T/F
7. All of the following viruses except _____ infect T cells, monocytes and macrophages.
a) HIV
b) SIV
c) FIV
d) BIV
8. Which of the following viruses is the only known vector-borne lentivirus?
a) FIV
b) CAEV
c) EIAV
d) JDV
9. Most naturally occurring SIV infections in African monkeys do not appear to result in
immunodeficiency disease but SIV arising via accidental or experimental
transmission in Asian macaques has resulted in several highly pathogenic SIV
strains for Asian macaques. T/F
10. Inoculation of chimpanzees with HIV-1 resulted in significant pathogenic changes
and AIDS induction. T/F
11. Genomic sequencing revealed SIVsmm as the origin of HIV2 in humans and SIVcpz
as the cross-species origin of HIV1 virus in humans. T/F
12. Name the animal model that offers the opportunity to experimentally infect human
tissues with HIV.
13. The MVV and CAEV models provide the opportunity to study genetic susceptibility
and host adaptation. T/F
14. FIV primarily targets CD4 cells. T/F
Answers:
1. c
2. d
3. T
4. b
5. F
6. T
7. d
8. c
9. T
10. F
11. T
12. SCID-human chimeric mice
13. T
14. T
Olivadoti et al. Murine Gammaherpesviruses 68: A Model for the Study of EpsteinBarr Virus Infections and Related Diseases, pp. 44-50
Summary: Epstein-Barr Virus (EBV) and Murine Gammaherpesvirus 68 (MHV68) are
two types of gammaherpesviruses (GHV) that infect humans and mice, respectively.
Gammaherpesviruses are species specific, so the study of EBV has been limited to
human studies. Since the identification of MHV68, studies have shown that MHV68
have many similarities with EBV in humans. Although there are differences, their
similarities outweigh their differences and create an opportunity for the use of MHV68 as
an in vivo model of EBV and other gammaherpesviruses that elicit debilitating behavioral
symptoms such as chronic fatigue and sleep problems. Both EBV and MHV68 cause a
life-long infection that is associated with various disease conditions. Both viruses
impede host immune processes and evade immune surveillance. Because of numerous
impediments and limitations to human GHV research, the availability of this in vivo
murine GHV infection will facilitate elucidations of mechanisms that underlie and link
virus reactivation, cytokine release, and clinical symptoms such as chronic fatigue
related to GHV infections.
Questions:
1. What characteristic of gammaherpesviruses has precluded using human GHVs in
animal models, making the study of their pathogenesis difficult?
a. Strong species specificity
b. Ability to form latent infections
c. Increased pathogenicity of human GHVs in other animal models
d. T lymphocyte suppression and increased secretion of interleukin 8
2. In 1980, MHV68 was identified as a natural pathogen of what two genus species?
a. Clethrionomys glareolus and Mus musculus
b. Mus musculus and Apodemus flavicollis
c. Apodemus flavicollis and Mesocricetus auratus
d. Clethrionomys glareolus and Apodemus flavicollis
3. Which of the following is not a type of gamma herpesvirus?
a. Epstein-Barr Virus
b. Herpesvirus papio
c. Herpesvirus tamarinus
d. Herpesvirus saimiri
Answers:
1. a - gammaherpesviruses are species specific
2. d - bank vole and wood mouse
3. c - H. tamarinus is an alpha herpesvirus
ORIGINAL RESEARCH
Mouse Models
Agca et al. Detection of Mouse Parvovirus in Mus musculus Gametes, Embryos,
and Ovarian Tissues by Polymerase Chain Reaction Assay, pp. 51-56
Task 1-Prevent, Diagnose, Control and Treat Disease
Species-Mouse (Mus musculus)
Summary: The authors collected germ cells and reproductive tissues from mice infected
with Mouse Parvovirus (MPV) and tested them for MPV using primary and nested
polymerase chain reaction (PCR). They found viral DNA (though not necessarily
infective virions) in a large proportion of sperm, oocytes, embryos and ovarian tissues.
Passing sperm samples through a Percoll gradient did not eliminate MPV. While
extensive washing of oocytes resulted in PCR negative for MPV; embryos that were
extensively washed still tested positive. MPV was detected in a higher number of
samples using nested PCR, rather than primary PCR. Though the authors did not
confirm in this report that the presence of viral DNA in infected germplasm will lead to
the production of MPV infected offspring, they urge caution when using infected (or
potentially infected) cells and tissues in Assisted Reproductive Technologies (ARTs)
such as embryo transfer, establishing embryonic stem cell lines, in vitro fertilization,
ovary transplantation, and intracytoplasmic sperm injection.
Questions:
1) What tissue is commonly taken to test for MPV infection using PCR?
2) What is the primary mechanism to eradicate MPV from enzootically infected
colonies?
3) True or False? Perforation of the protective membranes of oocytes with the
microinjection pipette impairs the cell's natural ability to prevent introduction of
infectious agents attached to the sperm plasma membrane during fertilization.
Answers:
1) Mesenteric lymph nodes
2) Rederivation by embyro transfer
3) True
Eaton et al. A Reproducible Scoring System for Quantification of Histologic
Lesions of Inflammatory Diseases in Mouse Gastric Epithelium, pp. 57-65
Summary: Histologic lesions in the gastrointestinal tract are difficult to quantify for the
following reasons. When samples are placed in formalin, the muscularis contracts
variably, resulting in distortion of the tissue, which complicates embedding, and
shrinkage in length, so that total length is not comparable between tissue preparations.
Semiquantitative systems are most commonly used for scoring gastric lesions due to
Helicobacter pylori. The severity of lesions in mice due to H. pylori varies according to
duration of infection, mouse strain, bacterial strain, and bacterial virulence factor
expression, and therefore scoring of histologic lesions is commonly employed.
In this study the authors sought to validate a reproducible scoring system for
Helicobacter pylori-associated gastric disease in mice. The scoring system is based on
quantification of the percentage of microscopic fields in which lesions are present, rather
than on subjective estimates of lesion severity.
Four investigators (readers) evaluated the slides and scored them. Histologic criteria
examined were: neutrophilic inflammation, gastritis sufficient to displace glands, and
epithelial metaplasia. Each microscopic field was scored only for presence or absence
of the lesion according to the agreed-upon definition, and the results were reported as
the percentage of fields affected on each slide. Linear regression analyses revealed
good agreement between investigators in scoring all the 3 histologic criteria examined.
Scoring the percentage of affected fields rather than subjectively estimating severity of
infiltrate resulted in good agreement among readers. The highest level of agreement
was in evaluation of neutrophilic infiltration. Metaplasia scores were the most variable
between readers, perhaps because it was the most difficult criterion to define as to what
constituted a metaplastic gland. This scoring system for H. pylori-associated gastritis in
mice is reproducible between readers and accurately reflects the disease status of the
mice.
Questions:
1. Helicobacter pylori is a human bacterial pathogen that is associated with:
a. Hepatitis, cholecystitis, colitis
b. Gastritis, peptic ulcer disease, gastric cancer
c. Typhlitis, chronic active hepatitis
d. Typhlocolitis, enteritis, ulceration in esophagus
2. ____________ was defined as any evidence of loss of parietal cells with
replacement by mucus-type cell.
a. Neutrophilic inflammation
b. Gastritis
c. Epithelial metaplasia
d. None of the above
3. All of the following statements are correct regarding the scoring system for H. pyloriassociated gastritis in mice except:
a. It is biologically and statistically valid for comparison of H. pylori-infected mice
b. Histologic lesions can be quantified reproducibly in conventionally prepared
sections of mouse stomach.
c. Quantification of gastric lesions was done subjectively.
d. It is reproducible between readers and accurately reflects the disease status of
the mice.
e. All of the above are correct
Answers:
1. b
2. c
3. c
Besselsen et al. Temporal Transmission Studies of Mouse Parvovirus 1 in BALB/c
and C.B-17/Icr-Prkdcscid Mice, pp. 66-73
Task 1: Prevent, Diagnose, Control, and Treat Disease
Primary Species: Mouse
This study was conducted to assess MPV shedding in feces from immune competent
BALB/c and immune deficient SCID mice. Authors also looked at effect of pregnancy
and lactation on fecal viral loads and transmission as well as biologic transmission of
MPV to naive mice and progeny of MPV-infected mice.
Authors obtained MPV1 from naturally infected mice during an epizootic in a commercial
barrier facility. Pregnant BALB/c mice, pregnant SCID mice, and weanling C3H mice
were obtained from Charles River, and were certified to be free from murine pathogens.
Standard rodent micro-isolator techniques (using hood) were used to handle mice. One
day old BALB/c and SCID pups were oronasally inoculated with MPV1. Pups were
weaned at 4 weeks age and evaluated over 24 weeks. Mice from each strain were
mated during this period. Fecal pellets were collected weekly for PCR analysis during
this period. C3H mice were used for sentinel exposure biweekly, and then euthanized
after a total of 5 weeks exposure for further organ PCR and serology analyses.
Data obtained indicated that SCID mice inoculated as neonates persistently shed high
levels of infectious virus that was transmitted well by both direct contact and by soiled
bedding exposure.
The BALB/c mice only shed high levels of virus in feces for 3 weeks post inoculation,
with seroconversion in sentinels exposed during the first 2 weeks post inoculation. After
this time period, there was only intermittent low levels of MPV DNA in feces of BALB/c
mice, lymphoid tissues of sentinel mice and progeny and breeding pairs.
Authors determined that pregnancy and lactation did not increase viral shedding in
BALB/c mice. However, there was enough MPV to induce infection in some progeny.
From this information, authors surmise that adaptive immune responses suppresses, but
does not eliminate MPV shedding. Though suppression can inhibit infection of weanling
and adult mice, some progeny may acquire infection.
Authors imply there is still much to learn with regards to MPV transmission. Sentinel
detection systems are problematic for this virus due to age susceptibility and adequacy
of viral load exposure.
Questions:
1. Which organs listed below best represent where one can detect viral DNA for MPV?
a. Stomach, spleen, kidney
b. Kidney, intestine, lung, lymph nodes
c. Spleen, liver, kidney
d. Heart, kidney, lymph nodes
2. Which is the reason for the great transmissibility of MPV among mice in research
facilities?
a. The virus is a common cell line contaminant, but it is easily eradicated.
b. The virus can produce persistent infections in mice and cell lines, but it is very
unstable in most environments.
c. The virus is highly stable in most environments, but difficult to infect most mice
strains.
d. The virus can be shed by feces, urine, and air, but not by direct contact.
e. The virus is stable, persistent, and difficult to eradicate.
3. Which strain is the most commonly detected strain of Mouse Parvovirus in
contemporary mouse colonies?
a. MPV1
b. MPV2
c. MPV3
d. MPV4
e. All of the above
4. Why is MPV detection so challenging?
Answers:
1. b
2. 3
3. a
4. Sentinel mice exposure to soiled bedding is used as a primary detection mode, and
this method is likely not optimal, as this article illustrates. There is a short period of
viral transmission post-infection in immune competent mice. A viral high does is
needed to induce productive infection.
Thomas III et al. Gender Influences Infectivity in C67BL/6 Mice Exposed to Mouse
Minute Virus, pp. 74-81
Purpose: The authors of this study experienced two natural outbreaks of MMV, 3.5 years
apart, at 2 separate facilities (one with micro-isolator caging and the other with ventilated
caging systems). Subsequently a study was designed to determine if cessation of
breeding in a colony leads to elimination of infection, to determine the length of time
MMV is shed in the feces, and the effectiveness of Swiss Webster dirty-bedding transfer
sentinel program in detecting MMV. C57B6 mice are naturally resistant to clinical signs
of MMV but mice from 2 sources (Tac and Cr) were selected for the study because
greater than 90% of the mice used at this institution are on a B6 background.
Study Design, Experimental Infections: 5 microliters (5x104 pfu) of an
immunosuppressive variant of MMV (MMVi) was obtained and used to experimentally
infect unanesthetized mice oronasally. Animals were bled on varying days, up to day 28
PI, and pooled feces for fecal PCR were colleted on varying days up to day 12 PI. Some
groups had PCR done on tissues.
Group 1: 4 week old male and female C57BL/6NCr or 4 week old male and female
C57BL/6NTac were infected with MMVi. The Cr males were negative on days 3 and 8
but on day 10, 12.5% were positive and 100% were positive by day 28. Similarly the
Tac males were seronegative on days 2, 9 and 13 but by day 16, 37.5% were positive
and by day 28, 100% were seropositive. The pooled fecal PCR for the Cr males was
negative for all days. The pooled fecal PCR results for the Tac mice was negative on all
days except day 9. For the females from both sources, the seroconversion rate never
reached 100% by day 28: Cr females had seroconversion in 56.2% by day 28 and the
Tac females had seroconversion of 62.5%. All pooled fecal PCR results were negative
on all days for all females from both sources.
Group 2: Four C57BL/6NCr lactating females were obtained and inoculated (the 21
pups were not inoculated). 16 live pups were euthanized on days 8, 14 and 28 and
blood, mesenteric LN, kidney and intestine were collected for PCR. The remaining
pups were weaned at 21 days. Dams were seronegative on day 3 and 10 but by day 16,
50% had seroconverted, and by day 28 100% had seroconverted. Blood samples from
random pups up to day 51 PI were negative, and PCR on euthanized pup tissues were
negative for days 8, 14, 28 PI.
Group 5: 3 configurations of Tac:SW for testing sentinel monitoring program. All 4 male
Tac:SW tested seropositive on days 48 and 170 while the females tested negative. No
research sentinels seroconverted before day 48.
Questions
1. MMV is:
a. Mouse Morbillivirus
b. Mouse Minute Virus
c. Mouse Mammary Virus
d. Mouse Mortality Virus
2. MMV is:
a. A nonenveloped, ssDNA virus of the family Parvoviridae
b. Noted for high rates of morbidity and mortality in all ages and genders of mice
c. Related to MPV
d. a & c only
3. T/F Because MMV and MPV are both parvoviruses, humoral immunity for one virus
is cross protective against the other?
4. What assays were performed in this study?
a. Enzyme-linked immunofluorescent assay (ELISA)
b. Hemagglutination-inhibition assay
c. PCR on mesenteric LN, kidney and intestine
d. Screen by ELISA, with positives confirmed by HIA and tissue PCR
5. T/F The fecal PCR assay is never effective for detecting animals shedding MMV.
6. What type of samples can be useful for PCR against MMV?
7. T/F Male and Female Swiss Webster mice had equivalent seroconversion rates
when used as sentinels in dirty bedding transfer programs?
Answers
1. b
2. d
3. F
4. d
5. F: Fecal PCR is most effective during or close to the acute phase of infection,
however catching mice during the fecal shedding phase is difficult, especially if there
are no clinical signs.
6. Fecal samples and tissues: mesenteric LN, kidney, intestine.
7. F: Female research sentinel mice were seronegative to MMV at most time points.
The research sentinel Tac:SW males seroconverted to MMV after transfer of dirty
bedding 3 times per week for 12 days but the female Tac:SW sentinels were
negative.
Smith et al. Induction of Pro- and Anti-inflammatory Molecules in a Mouse Model
of Pneumococcal Pneumonia after Influenza, pp. 82-89
Summary: Strep pneumonia is a leading cause of community acquired pneumonia and
sepsis in both adults and children. Bacterial pneumonia is also a frequent complication
of influenza, particularly in the elderly.
The purpose of the study is to characterize animal model and examine the
pathogenesis, pathology and immunology of pneumonia in the mouse model after
influenza virus infection. The study was conducted by challenging influenza virus
infected mice with three serotypes of pneumococccus. Influenza infected mice were
euthanized 24 hrs after secondary infection with S. pneumonia to determine the
pathology, bacterial culture, and levels of immune effectors or were followed by live
imaging system for development of pneumonia.
Elevated levels of both pro- and anti-inflammatory molecules including interleukins 6 and
10, macrophage inflammatory protein 1alpha, and chemokine KC were present in the
blood. High levels of these cytokines and chemokines as well as tumor necrosis factor
alpha, interleukin 1beta, and heme oxygenase 1 were present in the lungs, accompanied
by a massive influx of neutrophils. Mortality correlated with the development of
pneumonia and lung inflammation but not with bacteremia. This model has the potential
to help understand the pathogenesis of severe lung infections.
Question
True or False
1. Mortality after influenza virus infection is often due to secondary bacterial infection.
2. Mortality was correlated with development of pneumonia and lung inflammation that
bacteremia.
Answers
1. True
2. True
Smith et al. Reliability of Soiled Bedding Transfer for Detection of Mouse
Parvovirus and Mouse Hepatitis Virus, pp. 90-96
Summary
Introduction: Contemporary sentinel testing involves seroconversion of SPF mice by
exposure to soiled bedding. Protocols for doing so vary widely and the accuracy and
sensitivity of bedding transfer has not been assessed systematically for many agents or
for IVC. This study assessed the reliability of soiled bedding transfer of MPV (an
environmentally stable virus) and MHV (an environmentally unstable virus) by
determining the ability of bedding containing quantified amounts of MPV or MHV to elicit
seroconversion, the amount of soiled bedding with virus infected mice necessary to
transmit infection to sentinel mice at 3 time points and comparing these effects for both
IVC and static cages.
Results: Mice that were placed on bedding 4 hours after addition of virus spiked feces
showed dose dependent seroconversion. The longer the feces were allowed to sit before
introducing naïve sentinels had little effect on MPV but only induced infection with MHV
at the 4 hour mark. In static cages, index mice (inoculated with 30 X ID50 for MPV, and
300XID50 for MHV) seroconverted to both viruses and shed MPV in the feces for 2
weeks whereas the MHV infected mice shed for only one week. At 3 d, 1wk and 2wk
post inoculation, 25, 50, or 100ml of bedding were transferred to sentinel mouse cages.
In static cages, seroconversion for MPV occurred after 3 days only with the maximum
amount of bedding transferred, all volumes of bedding transferred after 1 week and this
declined at 2 weeks inoculation. For MHV at 3 days all mice seroconverted for all
transfer volumes but this tapered off at 1 week and 2 weeks. All sentinels placed in
direct contact seroconverted to MHV. In IVC caging, seroconversion of sentinels was
much greater for all time points for MPV, and yet still showed that this was greatest at 1
week. Seroconversion for MHV was 100% for all time points and bedding doses.
Mesenteric lymph nodes contained MPV DNA up to 18 weeks.
Discussion: MPV is stable in the environment for at least 7 days thus providing a wide
window for detection. MPV also resides in the LN's longer than the fecal shedding period
which could mean that the virus shedding could reoccur at times of immunocompromise.
MHV does not elicit seroconversion after 8 hrs, therefore bedding transfer are only
effective during the active shedding phase. The authors point out that the period of
detection is strain dependent and that shedding is strain dependent so this does not
necessarily apply to other scenarios. Seroconversion rates in the IVC were greater than
for static cages, which seem counterintuitive given that desiccation rates would be
greater in the IVC and would result in inactivation of the virus. The authors give two
possible rationales for this effect: the first is that because they used corn-cob bedding,
the ammonia levels would be lower in the IVC and there would be less inactivation of the
virus, and the second is that the increased air flow may have aerosolized virus particles.
For MPV the virus content of the soiled bedding pooled from several cages for exposure
to sentinels will often be low when cages are changed every week. Transfer of soiled
bedding is effective under optimal conditions for MHV and MPV but is less reliable in
contemporary colonies. The rate of seroconversion of sentinel mice was dependent on
volume of bedding. Soiled bedding transfer should be combined with other methods
such as contact methods and sub-set sampling. Frequent sampling is also
recommended.
Questions
1. MPV is an environmentally stable virus (True or False).
2. MPV does not last in the lymph nodes longer than the shedding period (True or
False).
3. In this study, transfer of soiled bedding was effective at producing seroconversion of
sentinels to MHV up to what time point:
a. 4 h
b. 3 d
c. 7 d
d. 14 d
4. MHV is an environmentally stable virus (True or False)
Answers
1. T
2. F
3. a
4. F
Amphibian Model
Godfrey et al. Newly Identified Mycobacterium Species in a Xenopus laevis
Colony, pp. 97-104
Task 1: Prevent, Diagnose, Control, and Treat Disease
Secondary Species: Xenopus spp.
SUMMARY: 3 colonies of Xenopus laevis frogs experienced multiple deaths over a 2
month period. Some were housed in static tanks and others were housed in a
recirculating system. All animals were fed frog brittle. The clinical presentation included:
coelomic distension, subcutaneous edema, and multiple raised skin lesions. Necropsy
revealed ascites and granulomatous lesions in the liver, spleen, and lung which
contained acid fast organisms. The organism was identified by PCR to be M. liflandi
which is commonly found in Xenopus tropicalis.
The Mycobacterium species in general are aerobic nonmotile acid fast organisms found
in soil, dust, vegetation, and water. The zoonotic species are: M. tuberculosis complex,
M. leprae,
M ulcerans, M. cheonae, M. xenopi, M. marinarum, and M. fortuitum. The
last 4 mentioned are carried by amphibians. M. ulcerans causes Buruli ulcers which are
painful and disfigurative. M. liflandi the organism described in this article is pathogenic
to Xenopus species but its zoonotic potential is not known at this time.
In light of the outbreak it was discovered that the standard method of sterilizing the tank
with ultra violet light at 225,000 uW-sec/cm2 was not sufficient. The treatment of the
condition entailed depopulation and treating the recirculation system with bleach at 200
ppm followed by a treatment with 1% Virkon. The system was also washed several times
with reverse osmosis water and 70% denatured alcohol (one time) before replacing with
new animals.
Questions
1) What is not true regarding the Mycobacterium species in general?
A) They are anaerobic
B) They are non motile
C) They are found on vegetation
D) All are not true.
2) What is not a clinical presentation of Xenopus frogs with M. liflandi?
A) Coelomic distension
B) SQ edema
C) Limb necrosis
D) Acute death
3) What is true regarding M. liflandi?
A) It is acid fast
B) Lethal to Xenopus species frogs
C) Zoonotic potential is unknown
D) All
Answers
1. A
2. C
3. D
Nonhuman Primate Models
D’Offay et al. Transmission Dynamics of Simian T-Lymphotrophic Virus Type 1
(STLV) in a Baboon Breeding Colony: Predominance of Female-to-female
Transmission, pp. 105-114
Species: Secondary
Task 1 Prevent, Diagnose, Control, Treat Disease
Summary: This article details an epidemiological study on the prevalence and modes of
transmission of STLV1 in a baboon breeding colony. Baboons in 4 different breeding
groups were evaluated over a 4 year period, and were screened for preexisting STLV1
and also new infections. PCR (specifically looking at tax gene) was the method used to
assess the infections, as it was proven more sensitive and consistent than using ELISA
or Western blot.
Common modes of transmission of HTLV in humans include mother-to-infant (highest
percentage), female-to-female, and male-to-female. Female-to-male transmissions
occur with much less frequency.
In the baboon populations, mother-to-infant
transmissions of STLV1 occurred much less frequently than with HTLV in the human
population. One hypothesis for this was the shorter time period that baboons nurse their
young. Female-to-female transmission of STLV1 was responsible for 73% of the new
STLV 1 infections. This was a surprising finding for the authors, and considered to be a
more important factor for transmitting disease because of conspecific fighting.
No questions or answers were provided with this summary.
Cohen et al. Seroprevalence of West Nile Virus in Nonhuman Primates as Related
to Mosquito Abundance at Two National Primate Research Centers, pp. 115-119
Summary: The purpose of this paper was to evaluate the seroprevalence of West Nile
Virus (WNV) in nonhuman primates (NHP) at the Yerkes Primate Center in Georgia, and
compare the results to the prevalence of WNV in NHP at the Tulane Primate Center in
Louisiana, which was determined in a previously published paper. WNV is a member of
the genus Flavivirus and is now endemic in the US. Blood was collected from 45 rhesus
macaques (Macaca mulatta) and 48 sooty mangabeys (Cercocebus atys) and antibody
titers to WNV were determined using serum neutralization and confirmed using a
hemagglutination-inhibition assay. In addition, mosquito surveillance data was obtained
in order to determine mosquito abundance and WNV infection prevalence in each of the
two areas (GA & LA). Results show that none of the rhesus macaques had antibodies to
WNV, and 3 of the 48 mangabeys were positive for WNV antibodies (6.3% prevalence).
This prevalence data is quite different from the earlier published Tulane data, which
found that the prevalence rate of WNV in NHP at TNPRC is relatively high at 36%. The
ratio of seroprevalences in the TNPRC and Yerkes primate populations was similar to
the ratio of WNV incidences in people in LA and GA in 2002 and 2004. The differences
in the exposure of NHP to WNV between these two regions is consistent with the fact
that the abundance of WNV-infectious mammal-biting mosquitoes was 23 times higher
at TNPRC than at Yerkes in 2003 and 33 times higher in 2004.
Questions:
1. Name the genus and species of the rhesus macaque.
2. Name the genus and species of the sooty mangabey.
3. WNV is a member of what genus?
4. Why is the prevalence of WNV suspected to be higher at the TNPRC in LA
compared to Yerkes in GA?
Answers:
1. Macaca mulatta
2. Cercocebus atys
3. Flavivirus
4. The abundance of WNV-infectious mammal-biting mosquitoes is significantly higher
at TNPRC than at Yerkes.
CASE STUDIES
Mitsunaga et al. Changes in the Titer of Anti-B Virus Antibody in Captive
Macaques (Macaca fuscata, M. mulatta, M. fascicularis), pp. 120-124
Task 1 - Prevent, Diagnose, Control, and Treat Disease
Species - macaques, primary
SUMMARY: This article examines 3 cases of changes in anti-B virus antibody in relation
to environmental stresses including transport, transfer and during breeding season. The
B virus (Herpesvirus simiae, Cercopithecine herpesvirus 1, BV) is an alpha-herpesvirus
and only causes mild or asymptomatic infection in its natural macaque host. However,
BV in humans usually causes fatal central nervous system infections, and is classified as
a biosafety level-4 pathogen. In case 1, 2 BV seropositive male cynomolgus macaques
were transported via air freight. Two weeks later, one monkey had a >15 fold increase
in level of anti-BV. In case 2, four BV-seropositive male rhesus macaques were moved
from outdoor group caging to individual indoor caging. After one week, one of the 4 had
a 6X increase in anti-BV titers. In case 3, 2 out of 3 male Japanese macaques had 2X
increases in their anti-BV titers during breeding season. 4 female macaques had higher
anti-BV titers than the males, but were stable through the year.
Since BV establishes latent infection in macaques, the factors inducing BV reactivation
and shedding are of great importance. This case study examined the potential impact of
transport and housing changes on titers. An increase in anti-BV IgG levels may be a
sign of reactivation of virus. Stress can be a trigger to reactivation. Further, breeding
changes such as increased testosterone and glucocorticoids may be potential factors in
reactivating BV. Shedding was not examined in this study. The findings suggest that
BV reactivation may be a fairly frequent event in monkeys during common management
practices.
QUESTIONS: True/ False
1. The B virus establishes latent infection in macaques.
2. Herpesviruses may be reactivated from latent infections but do not shed when
reactivated.
3. Common housing changes may cause 2- 15X increases in anti-BV titers in a
percentage of macaques.
ANSWERS
1. T
2. F
3. T
Sasseville et al. Naturally Occurring Tyzzer’s Disease in Cotton-top Tamarins
(Sanguinus Oedipus), pp. 125-127
Summary: Cotton-top Tamarins are a small New World primate species that serve as an
important natural model for inflammatory bowel disease, colon carcinoma, and hepatitis
C infection. These animals exhibit a high rate of neonatal mortality due for several
reasons and those that survive past 4 months of age have a high rate of idiopathic colitis
with progression to colonic adenocarcinoma. The authors of this article describe 2
cases of captive-reared male CTTs that died of severe colitis.
Histopathologic results for the 2 animals showed marked diffuse inflammation of the
colon and cecum, characterized by an infiltrate of mononuclear cells, neutrophils, and
scattered eosinophis from the base of colonic and cecal crypts through the muscularis
mucosae, submucosa, and tunica muscularis to the serosa. In addition to typhlocolitis,
both animals had hepatic and myocardial necrosis and inflammation. Because these
three lesions are suggestive of Tyzzer’s Disease, sections were stained for C. piliforme.
All three tissues from both animals had abundant silver-positive, large slender rods with
hepatocytes at the edge of the necrotic lesions; along the long axis within myofibers in
the cecal and colonic tunica muscularis and within myocytes at the edge of the heart
lesions. Therefore the diagnosis of Tyzzer’s Disease was made.
Spontaneous Tyzzer’s disease occurs in several laboratory and domestic animal species
an din wild animals, including marsupials and psittaciforms. Cases of Tyzzer’s in
humans and nonhuman primates are extremely rare, with only 1 report each in a patient
infected with HIV-1 and in Old World nonhuman primates.
With the CTTs raised in captivity at the New England Primate Research Center,
parental rejection commonly occurs as with the 2 animals in this case. It is thought that
transplacental immunoglobulin transfer may be ineffective in CTTs which may result in
increased susceptibility to infectious diseases. The source of infection is unknown but
thought to be from environmental contamination.
The conclusion is that Tyzzer’s Disease should be included in the differential list for
colitis in CTTs and search for the concurrent hepatic and cardiac lesion should be
conducted.
Questions:
1) What are the three lesions that are consistent with a diagnosis of Tyzzer’s Disease?
2) The organism that causes Tyzzer’s Disease is Staphylococcus Aureus. T or F
3) Cotton Top Tamarins are used as a model of what type of disease?
4) Tyzzer’s Disease was first recognized in what species?
Answers:
1) Necrosis in cecum-colon, liver and heart.
2) F. It is caused by C. piliforme (previously called Bacillus piliformis)
3) Inflammatory bowel disease, colon carcinoma and Hepatitis C virus infection
4) Japanese waltzing mice.