Services 2017-08-30T23:07:01+00:00

PDL Services

Consultation Services

One of the distinctive features of PDL is our ability to work with clients in understanding and interpreting laboratory data as it relates to their NHP colony. Senior staff can provide expertise in experimental design, assay development, interpretation of diagnostic testing, case and colony management.

In addition, we have a strong record of technology transfer having provided training and protocols to numerous visiting scientists who have then successfully implemented assays in their own laboratories.

Some of PDL’s ongoing research interests include the discovery and understanding of new emerging infectious agents, and the application of new, state of the art laboratory diagnostic methodologies. Please contact us to discuss your specific interests.

Reagents

PDL maintains a large archive of appropriately preserved and stored sera, plasma, DNA, cells and other NHP body fluids and tissues. These materials have been characterized for their reactivity to various agents including SRV, SIV, STLV, SFV, Herpes B, RhCMV, TB, and RRV. We also maintain stocks of virus isolates, cells, proteins, and nucleic acids for similar agents. PDL routinely uses these materials for quality control, process documentation, proficiency, training, and assay optimization and validation.

As sample volume permits, these reagents are available on a recharge basis for sharing with other investigators. Please contact us to discuss your specific interests.

Routine Testing

ABSCN-5 Includes: SRV, SIV, STLV, Herpes B Surrogate Marker, Measles

ABSCN-8 Includes: SRV, SIV, STLV, Herpes B Surrogate Marker, Measles, RhCMV, SFV, RRV

ABSCN-Custom Includes: ABSCN-8 and addtional agents by request (SV40, LCV, custom).

The Antibody Screen panels utilize Multiplex Microbead ImmunoAssays to detect the host’s humoral immune response to various infectious agents. The panels simultaneously detect virus-specific antibodies in serum, plasma or other body fluids. The antibody screen assays are run on a Luminex platform using panels of microbeads coated with purified antigens. Each antigen bead has uniquely identifiable spectral signature.

Microbeads are incubated with diluted serum samples so that any specific antibodies present will bind to the matching antigen coated beads. The beads are washed, and reacted with phycoerythrin-conjugated detection reagents. After additional washing, independent gated events for each bead are counted and analyzed in a Luminex cytometer.

The median fluorescence intensity (MFI) of antigen-coated bead sets is compared to control coated beads in each serum. These assays were designed as screening assays with optimal sensitivity and require confirmatory testing by more specific methods.

The minimum sample requirement is 1 ml of serum or plasma sent frozen.

Antibody Screen (ABSCN) results can be confirmed by Western Blot. This method allows visualization of antibody reactivity specific to viral (and non-viral) proteins as separated by molecular weight.

Individual WB’s available for: SRV1, SRV2, SRV5, SIV and STLV. In the WB assay, if specific antibody is present in the serum sample it will bind to the specific antigen bands on the PVDF or Nitrocellulose strip. The presence of antigen-bound antibody is detected by the addition enzyme conjugated detector antibody.

Substrate is added that will yield a color change where the enzyme has been bound (positive for antibody). The minimum sample requirement is 1 ml of serum or plasma sent frozen.

Antibody Screen (ABSCN) results can be confirmed by IFA.

Individual IFA’s available for: Measles, SFV, RRV, SV40, LCV and Varicella.

The Immunofluorescence assay (IFA) can be used to detect antibodies to specific virus in infected cells coated on microscope slides. In this assay, test and control serum samples are reacted on the test antigen infected and uninfected control cells.

If specific antibody is present in the test sera, it will bind to the test antigen infected and not the uninfected control cells. The specific antibody – test antigen binding is detected by binding a secondary, fluorescent conjugated detector antibody. The signal is observed by fluorescence-microscopy.

The minimum sample requirement is 1 ml of serum or plasma sent frozen.

Individual PCR available for: SRV (1-5), SFV, STLV, RRV and SIV.

PCR assays are used to detect viral nucleic acids (DNA, RNA, proviral DNA) in various body fluids, whole blood or tissue. This assay allows us to detect and amplify very small quantities of viral or proviral nucleic acid. The appropriate nucleic acid is extracted from blood or tissue and then amplified in the PCR assay.

All of our current assays use Taqman1 or Real Time PCR. Probes and primers have been designed to amplify specific viral gene sequences.

In addition, probe and primers to amplify the housekeeping gene oncostatin M (OSM) which encodes a cytokine are run in each reaction. The OSM gene is diploid in all primate cells allowing for cell quantification. The OSM signal is used to assure that we obtained amplifiable DNA for all samples.

Although for many viruses a PCR result is diagnostic, for SRV, both PCR and serology are necessary to rule out infection.

The minimum sample requirement is 3 ml of heparinized whole blood sent at room temperature to arrive within 24 hours of collection.

In vitro assays for Interferon Gamma (IFN-g) response to tuberculin antigens represent a refinement of the lymphocyte stimulation assay. As soon as possible following collection, aliquots of heparinized whole blood are stimulated with control and TB antigens in pre-loaded tubes. After 24 hours incubation, the concentration of IFN-g in the supernatant plasma of each aliquot is determined by enzyme immunoassay.

In experimental primate infection models, cell mediated immune responses are detectable in vitro 2 to 4 weeks after infection. IFN-g is a critical cytokine in the cell mediated immune response to tuberculin antigens, including the DTH response measured by the TST, and in the host immune response to infections with tuberculous mycobacteria. Primagam (Prionics, USA Inc, La Vista, NE) is a commercial version of this assay using bovine and avian purified protein derivatives (PPD) as antigens.

Please contact us to arrange to receive instructions and pre-loaded stimulation tubes to be filled, incubated and processed on-site immediately following collection.

Custom/Other (IHC, ISH, etc.)

Please call for more information about PDL offered ELISA/ EIA’s.

The presence or absence of antibodies in serum or plasma can be determined qualitatively by Enzyme Immuno Assay. In these assays, microtiter plate wells are coated with antigen (typically purified viral lysate). Diluted serum is incubated in the wells. If specific antibody is present it will bind to the antigen. The enzyme conjugated detector antibody is added next. Finally a chromogen reagent is added. The development of color in the wells signifies the presence of specific antibody.

The minimum sample requirement is 1 ml of serum or plasma sent frozen.

Immunohistochemistry, or IHC, refers to the process of visualizing antigens (e.g. proteins) in cells of a tissue section. Specific antibodies are used to bind specifically to antigens in biological tissues.

Immunohistochemical staining is widely used in the diagnosis of abnormal cells such as those found in cancerous tumors. Specific molecular markers are characteristic of particular cellular events such as proliferation or cell death (apoptosis). Virus proteins can be visualized in specific tissues and specific cell compartments using this technique. This is particularly useful in cases where suspicion of the infection doesn’t occur until after euthanasia and typical blood and plasma samples are not available.

Immunohistochemistry stains thin slices of formaldehyde-fixed paraffin-embedded (ffpe) tissues on glass slides. Specific antibody (poly- or monoclonal) targeted to the antigen is incubated with the tissue. A secondary antibody, conjugated to an enzyme (e.g. peroxidase) or fluorescent tag is then used to visualize the binding. The enzyme/conjugate catalyzes a reaction that results in colored deposition in the tissue. PDL offers SRV IHC using a monoclonal gp20 antibody that cross-reacts with serotypes 1-5. Please inquire about custom IHC for other pathogens.

In situ Hybridization, or ISH, refers to the process of visualizing specific nucleotide sequences in tissue sections. The tissue is treated to encourage hybridization. A conjugated probe is used to specifically hybridize to the sequence of choice and then visualized. We are currently validating ISH for SRV and other pathogens. This assay is offered on a custom basis.

Other assays are available or can be developed to answer specific research or diagnostic questions. The charge is actual labor plus cost of reagents. Please contact us to discuss.

Some examples include:

  • Virus isolation for SIV, SRV, SFV, RhCMV and RRV
  • SRV Immunohistochemistry (IHC)
  • SRV In-situ Hybridization (ISH)
  • PCR typing for SRV 1-5
  • Quantitative PCR
  • RNA PCR
  • Quantitative antibody titers Cytokine /Chemokine Detection and Quantitation Assays (EIA and MIA) including IL1b, IL1Ra, IL2, IL4, IL5, IL6, IL8, IL10, IL12 (p70), IL13, IL15, IL17, IL18, sCD40L, G-CSF, GM-CSF, IFNg, MCP-1, MIP-1a, MIP-1b, TGFa, TNFa, VEGF, Rantes, TGF-1
  • Custom culturing and processing.

PDL offers testing for the following Pathogens

Infected animals with active TB may show no overt signs of disease for weeks or months, during which time they can transmit infection to other colony animals. Animals with latent TB are not infectious and may appear healthy for years, but eventual reactivation of latent TB can result in secondary transmission and outbreaks of disease in established colonies.

Reactivation of latent infections that were not detected using traditional screening methods during primary quarantine is emerging as an important factor in the epidemiology of TB in nonhuman primates. In experimental primate infection models, cell mediated immune responses are detectable in vitro 2 to 4 weeks after infection. IFN-g is a critical cytokine in the cell mediated immune response to tuberculin antigens, including the DTH response measured by the TST, and in the host immune response to infections with tuberculous mycobacteria.

Primagam (Prionics, USA Inc, La Vista, NE) is a commercial in vitro assay for Interferon Gamma (IFN-g) response to tuberculin antigens. It represents a refinement of the lymphocyte stimulation assay. This assay uses bovine and avian purified protein derivatives (PPD) as antigens. We have modified the commercial assay to include additional antigens, incubation conditions, and cytokine levels. Whole blood is stimulated with control and TB antigens. After 24 hours incubation, the concentration of IFN-g in the supernatant plasma of each aliquot is determined by enzyme immunoassay.

Please contact us to arrange for instructions and materials prior to collecting samples.

Work on additional assays is ongoing. Please contact us for the latest updates.

The exogenous simian type D retroviruses (SRV) are a group of closely related viruses that have been isolated from Asian monkeys. These isolates are related to the prototypic type D retrovirus, Mason-Pfizer monkey virus (MPMV).

SRV has been isolated from many macaque species including rhesus, cynomolgus, and pig-tailed monkeys. Although all macaque species appear susceptible to all serotypes, some general serotype-host species associations have been recognized. Cynomolgus and pig-tailed macaques are predominantly infected with serotype 2, while in rhesus SRV-1 is the predominant serotype. Prevalence of SRV infection in captive populations of macaques is variable. Geographic origin of animals, as well as management and husbandry practices, are factors influencing the level of SRV endemicity in macaque populations.

SRV has a broad cellular tropism, including both lymphoid and non-lymphoid tissues. SRV can be demonstrated in many tissues and organs and has been isolated from many body fluids, including saliva, urine, blood, lachrymal secretions, cerebrospinal fluid, and breast milk. Transmission of SRV occurs horizontally, either through direct contact between infected and susceptible animals, or indirectly through contact with contaminated instruments or equipment. A major mode of transmission is via contact with virus shed in saliva, either during mutual grooming or aggressive interactions involving biting and scratching. Transplacental transmission has been documented.

SRV can elicit a broad spectrum of clinical and pathologic manifestations, ranging from subclinical carriers to rapidly fatal immunosuppressive disease. Common clinical findings in SRV-infected macaques include diarrhea, weight loss, splenomegaly, lymphadenopathy, anemia, neutropenia, lymphopenia, and occasional neoplastic disease, including cutaneous fibrosarcoma and retroperitoneal fibromatosis. B-cell lymphomas have been reported in Cynomolgus macaques.

A significant number of SRV-infected macaques are seronegative. Parallel testing for both SRV antibody and virus (PCR), at more than one time point is required to accurately identify all infected animals.

Testing for proviral DNA is available by Taqman real-time PCR which targets the env gene. Quantitation and typing may also be available by arrangement. Please contact us. Antibody screening is included in our Antibody Screening Panels (AbScn5, 8, Custom). Confirmatory testing by Western blot prepared with SRV 1,2, or 5 virus is available.

Simian T-cell Lymphotropic Virus (STLV) is a C-type member of the oncornavirus subgroup of retroviruses. It is endemic in nonhuman primates originating in Africa and Asia.

Reports of STLV seroprevalence vary from 0-80% depending on the test algorithm used; and the species, age, and sex of animals tested. Estimates in captive populations of macaques housed in the USA have ranged from 3-12%. An apparent long interval (years) to seroconversion has been observed in long-term follow-up of STLV serongeative colonies. Although there are reports of etiologic links between STLV and malignant lymphomas and lymphoproliferative diseases in some primate species most STLV infection is unapparent, as the monkeys remain clinically healthy and asymptomatic. However, altered cytokine profiles have been reported.

There is a 90-95% homology between the structural proteins of STLV and Human T-cell Lymphotropic virus, type 1 (HTLV-1), the etiologic agent of adult t-cell leukemia/lymphoma and the progressive neurologic disease, Spastic Paraparesis.

Testing for proviral DNA is available by Taqman real-time PCR which targets the tax gene. Antibody screening using STLV cross reactive HTLV purified virus antigens is included in our Antibody Screening Panels (AbScn5, 8, Custom). Confirmatory testing by Western blot prepared with HTLV virus is also available.

Simian immunodeficiency viruses (SIV) are a group of genetically related viruses belonging to the lentivirus subgroup of retroviruses. The natural hosts of SIV include many species of African monkeys and the chimpanzee. Prevalence of infection in various populations of captive and wild African primates may exceed 50%. Infection in these natural host species, however, is almost always subclinical.

Asian macaques are susceptible to SIV infection, but naturally acquired (i.e. non-experimental) infections in macaques are an artifact of captivity, the result of cross-species transmission from African species through direct contact with infected animals or their tissues or body fluids. SIV is not a naturally occurring infection of wild macaques in their countries of origin. Various strains of SIV are highly pathogenic in macaques, and produce a severe immune deficiency disease. SIV rarely causes overt disease or histologic lesions in natural host species.

Testing for proviral DNA is available by Taqman real-time PCR which targets the SIVmac 251/239 gag gene. Antibody screening is included in our Antibody Screening Panels (AbScn5,8,Custom). Confirmatory testing by Western blot prepared with SIVmac virus is also available.

Cercopithecine Herpesvirus (Herpes B, or “B virus”) is highly prevalent in most macaque populations. It can be transmitted through oral or sexual contact. Prevalence of up to 90% has been reported in natural adult populations.

Although a persistent infection, it is characterized by latent periods with occasional viremic periods. While not usually a clinical problem for macaques, the potential for zoonotic transmission causing severe and even fatal infections in humans makes it a significant biohazard concern. The virus can only be propagated under biohazard containment conditions that make it difficult to produce antigen for testing.

Fortunately, a number of investigators have documented good cross reactivity between this virus and human and nonhuman primate alphaherpes, which can be used for diagnostic testing.

Antibody screening for Herpes B using is included in our Antibody Screening Panel (AbScn). 2 different purified Herpes Virus Papio type 2 (HVP2) antigens are used as surrogate makers in our MMIA assay. HVP2 is a herpes virus of baboons which is genetically and antigenically closely related to B Virus. We have recently validated an MIA using inactivated B Virus as a confirmatory test.

Measles virus is a highly infective paramyxovirus of the genus Morbillivirus. Human and nonhuman primates are the only known hosts for MV infection. Sporadic measles epizootics with high morbidity and mortality have been reported in macaque colonies. Clinical symptoms in nonhuman primates are similar to those seen in infected humans including maculopapular rash, conjuctivitis/blepharitis, coryza, lymphadeonpathy, pyrexia, cough, anorexia, and malaise.

Measles virus is immunosuppressive, causing transient humoral and cell-mediated immune dysfunction which can predispose infected individuals to a variety of secondary complications including septicemia, abortion, metritis, pneumonia, severe dysentery, and disseminated intravascular coagulopathies. Since transmission from humans to nonhuman primates is well documented, in addition to vaccination of nonhuman primates prevention strategies include human heath surveillance, vaccination, and restricted access.

Antibody screening using a recombinant nucleocapsid protein target antigen is included in our Antibody Screening Panel (AbScn-5); and an indirect Immunofluorescent Assay (IFA) is available for confirmatory testing. A neutralizing antibody titer assay using green fluorescent protein labeled virus is also available by request. Please contact us for details.

Foamy viruses are highly prevalent in virtually all species of nonhuman primates, approaching 100% infected in adult animals in many populations. At least 12 serotypes or genetic variants are currently recognized. SFV appears to be nonpathogenic in all primate hosts.

SFV establishes latent infection in many different tissues, while replication appears to be restricted to the mucosa of the naso- and oral- pharynx. SFV appears to produce no lesions in vivo; however reactivation of latent virus in cultures of primary cells from nonhuman primates produces a highly cytolytic infection with rapid cell death. This has significance for studies requiring the growth or maintenance of primary cultures, and transplant studies.

Little is known regarding the more subtle effects of SFV infection that might adversely affect primate studies. Unlike other retrovirus infections, SFV appears not to induce an increased interferon-g response. SFV infection does, however, alter cell surface markers, inducing increased MHC class I expression.

Testing for proviral DNA is available by Taqman real-time PCR which targets the SFV pol gene. Antibody screening using SFV purified virus is included in our Antibody Screening Panels (AbScn 8 and custom). Confirmatory testing by IFA is available.

Rhesus Cytomegalovirus (RhCMV) is a beta Herpesvirus which can persistently infect macaques. Most are seropositive by 1 year of age. Although infection is asymptomatic in the immunocompetent host, virus is shed periodically. RhCMV is an opportunistic pathogen which can lead to severe and even fatal disease in immune compromised monkeys, such as those co-infected with SIV or SRV, or immunosuppressed for organ transplantation.

Antibody screening for RhCMV using purified virus is included in our Antibody Screening Panels (ABSCN-8 and ABSCN-custom).

The Rhadinovirus genus of gamma-2 herpesviruses is divided into two subgroups, RV1 and RV2, based on genomic sequence comparisons. Rhesus rhadinovirus (RRV) is a member of the RV2 subgroup and naturally infects rhesus macaques. The ability to establish both lytic and latent infections, a hallmark of the Herpesviridae family, is observed in rhadinovirus infections. RRV isolates have shown significant sequence similarity to Kaposi’s Sarcoma Herpes Virus (KSHV) and Retroperitoneal Fibromatosis Herpes Virus (RFHV).

Rhadinovirus infections are generally subclinical in immune competent natural hosts and overt disease is thought to arise only when hosts are immune-compromised. Experimental co-infection of rhesus macaques with SIV and RRV results in a lymphoproliferative disease resembling multicentric Castleman’s Disease; however variations in disease outcome have been reported.

RFHV has been reported to be associated with retroperitoneal fibromatosis in SRV2-infected macaques. Our studies indicate that both RRV and RFHV are highly endemic in socially-housed rhesus macaques, and infection occurs at an early age in a pattern similar to that observed for the betaherpesvirus, rhesus cytomegalovirus.

Testing for RRV is offered by Taqman Real Time PCR amplification and detection of the polymerase gene. Primers for Taqman Real Time PCR amplification and detection of RFHV have been developed and the assay is currently in validation. Please contact us for current status.

Serological testing for RRV is available by Antibody Screen (ABSCN-8 and ABSCN-custom). Confirmatory antibody testing is available by IFA.

Simian lymphocryptovirus is an EBV-like gamma-Herpesvirus which persistently infects most macaques. It is highly prevalent in most colonies. LCV has been implicated in the etiology of lymphoproliferative disease and B-cell lymphoma in rhesus macaques immunocompromised due to SIV infection or chemical immunosuppression for whole-organ transplant studies.

Testing is performed using an EBV Viral Capsid Antigen Immunofluorescence Assay as a surrogate for LCV. LCV antibody testing is also being developed as part of the custom Antibody Screen Panel. Please contact us for information and scheduling.

Simian virus 40 is a member of the polyomavirus subgroup of Papovaviridae. SV40 is a common persistent infection in macaques and Green monkeys. It can cause lesions on the kidney and brain in immunodeficient monkeys. It is an opportunistic virus causing progressive multifocal leukoencephalopathy in SIV-infected macaques, and has also been shown to cause mesotheliomas (rare lung tumors), non-Hodgkin’s lymphomas, interstitial nephritis and multifocal leukoencephalopathy in lab animals.

Serological testing for SV40 can be included in the custom Antibody Screen (ABSCN-custom). Confirmatory antibody testing is also available by IFA.

Simian Varicella Virus (SVV) infection in nonhuman primates is similar to Varicella Zoster Virus (VZV) in humans. Both viruses share common clinical, pathological, immunological and virological characteristics in their respective hosts, causing a varicella-like disease characterized by fever and a vesicular skin rash on the face, torso, and extremities. Acute disease can progress to latency and potential reactivation.

Reported natural routes of transmission include inhalation of airborne virus and direct contact with skin lesions. Epizootics ranging from mild symptoms to severe morbidity and mortality have been reported.

Assays to detect SVV antibody are available in our Custom Panel. Please contact PDL for more information.

Other assays are available or can be developed to answer specific research or diagnostic questions. The charge is actual labor plus cost of reagents. Please contact us to discuss.

Some examples include:

  • Virus isolation for SIV, SRV, SFV, RhCMV and RRV
  • SRV Immunohistochemistry (IHC)
  • SRV In-situ Hybridization (ISH)
  • PCR typing for SRV 1-5
  • Quantitative PCR
  • RNA PCR
  • Quantitative antibody titers Cytokine /Chemokine Detection and Quantitation Assays (EIA and MIA) including IL1b, IL1Ra, IL2, IL4, IL5, IL6, IL8, IL10, IL12 (p70), IL13, IL15, IL17, IL18, sCD40L, G-CSF, GM-CSF, IFNg, MCP-1, MIP-1a, MIP-1b, TGFa, TNFa, VEGF, Rantes, TGF-1
  • Custom culturing and processing.