Diagnostic Testing Lab – Department of Clinical Sciences
Vector Borne Disease Diagnostics
Lab Contact: 919.513.8279
We are currently working under reduced operations with limited staff in response to COVID-19. Please note that our laboratory is still open and receiving samples, but the University could be subject to closure with very little notice. Please continue to check our website for updates before shipping samples to our laboratory. Test turnaround times may be affected by Reduced Operations, but we are working to get results out as quickly as possible. We appreciate your patience.
Due to insufficient Babesia canis IFA slides, we are currently not offering B. canis IFA serology testing until we are able to replenish our supply. This will affect individual Babesia serology requests, Canine Serology Tick Panels and Canine Comprehensive Panels. We will continue to offer B. gibsoni IFA testing, which can cross react with antibodies to B. canis.
Data collected from dogs screened by both B. gibsoni and B. canis IFA between 2010 – 2016, showed that 68.5% of dogs with anti-B gibsoni antibodies also had antibodies against B canis, likely representing cross reactivity between the two species.
If you are suspicious of a B. canis infection, please consider requesting a Babesia PCR test.
We apologize for any inconvenience this may cause. We will provide an update on our website once we are able to anticipate when slides will be available again. It will likely be a few months before slides are replenished. Please don’t hesitate to contact us with questions or concerns.
You are invited by researchers at The University of Georgia, Hollins University, Duke University, Clemson University, and the University of Rhode Island to participate in a research study entitled “Investigating COVID-19 impacts on the epidemiology of tick-borne diseases in people and pets.
Any person who is 18 or older and a resident of the United States or Canada is eligible to participate. Please click the link if you elect to participate in this survey.
FAQs for VBDDL Sample Submission and Testing
Preliminary results will now only be given via email. If you have any inquiries on current test status or would like an update on preliminary results, please contact us from your clinic email address. An automated results report will also be sent out via email once all testing is finalized.
NCSU/CVM, VBDDL, RM 462A
1060 William Moore Drive Raleigh, NC 27607
Phone #: 919-513-8279
We do not recommend shipping samples with a delivery date longer than two days from ship date. Samples cannot be shipped via priority mail or through the US Postal Service since there is a significant delay in package delivery to our location. Samples should be shipped with an icepack to keep samples cool. Samples must be properly labeled with the patient’s name and sample type and must have matching submission paperwork enclosed with the sample. If sending samples from multiple patients, it is advised that a packing list also be included with the shipment (indicating the patient names and specimen types of all samples in package). Submitted samples must always satisfy federal shipping regulations in the event of breakage/leakage, (i.e. wrap in absorbent towels or bubble wrap and place in sealed plastic bag within a Styrofoam or cardboard box). It is helpful to seal the tops of tubes with parafilm when packaging the sample to help decrease the chance of leakage during shipment.
Unfortunately, our laboratory does not have any special shipping promotions or pre-paid shipping labels available. If shipping samples to us from another country, please contact us via email so that we can further assist you in ensuring that you have importation permits from our lab.
Our laboratory is closed during the weekend. Any blood samples collected on Friday or Saturday can be held in the refrigerator and shipped on Monday. Samples may be stored in the refrigerator for up to two weeks (this is especially useful if trying to save on shipping costs by batching multiple samples in a single shipment). Our laboratory is closed on most federal holidays. North Carolina State University also has a mandatory closing for energy conservation during the winter holiday. Please check our website for details on our hours of operation.
No preliminary information is needed when creating an account with us. Our accounts are associated by the clinic address and phone number. Please make sure to include clinic name, phone number, and address on the submission paperwork. If there is no existing account for your clinic, a new account will be created for you when we receive your samples and paperwork. Accounts can only be created for veterinary clients and cannot be created for owners as the results must be interpreted by a licensed veterinarian.
Invoices are sent out monthly and any billing correspondence will be sent to the clinic mailing address found on the submission form. Please do not send payment for testing with submission paperwork. Any billing questions can be directed to our Diagnostic Lab Accountant, Denise Crowell, at 919-513-6305 or email@example.com
We take special care in making sure that our testing is thorough and results are reported accurately. Our IFA testing is currently taking 10-15 business days to complete and PCR testing typically takes 3-5 business days for completion due to large sample submission volumes. Our turn-around time can also vary in cases where results need to be confirmed or repeated. We are happy to give an update on preliminary results at any time. Please contact us by email at firstname.lastname@example.org with any preliminary result inquiries. A final result report is sent out via automated email once all testing is completed.
We recommend sending EDTA-anticoagulated whole blood (lavender top tube) and serum (serum separating tube), red top tube, or tiger top tube) with each test submission. There is no cost associated with holding samples for potential additional add-on tests. We request that you send at least 2mL of each sample type, but tests can be run with as little as 0.5mL volume. The 0.5mL volume does not allow for samples to be banked for troubleshooting and/or repeated testing. Our serology IFA tests and the SNAP®4DX®Plus are run with serum and PCR tests are run with DNA extracted from EDTA whole blood. We recommend that both serum and EDTA whole blood be submitted regardless of which testing is requested. It is useful to have pre-treatment samples banked back for acute and convalescent pairing of serology tests and for testing of pre-antibiotic samples by PCR. Samples are banked back for at least two years and additional testing can be requested at any time.
Any sample type can be submitted for PCR testing. An additional charge of $75 must be added to cover the costs of the special handling required to extract DNA from fresh/frozen tissue or from formalin-fixed paraffin embedded samples. Fresh (frozen or refrigerated) tissues are the preferred sample type for non-blood submissions. Fresh tissue samples can be stored in an EDTA or no additive tube with sterile saline to prevent the tissue from drying out. Tissue samples stored in formalin solution are not recommended as prolonged exposure to formalin causes DNA crosslinking which can result in false negative PCR results. Vascular tissue samples such as spleen or liver are standard tissue types for PCR testing, but any type of tissue can be tested. We usually recommend testing tissues that are showing signs of pathology. Tissues can be pooled together and tested as a single sample or can be tested separately (please indicate on request form how you would like samples tested if sending multiple tissue types). Formalin-fixed or parafin embedded samples can be submitted by sending the whole tissue block or by sending pre-prepared scrolls (please send at least 10 scrolls if not sending a whole block).
Bone marrow aspirates are also commonly submitted samples for PCR testing and generally do not require a $75 tissue extraction fee. Please make sure to send sample in a lavender top tube and clearly indicate on the submission form and the sample tube that the sample is from bone marrow so it is not confused with EDTA whole blood.
Lymph node aspirates can be useful samples for diagnosing vector-borne pathogens by PCR. Testing lymph node tissue is can increase the sensitivity when submitting samples for Leishmania PCR testing. You may also add lymph node aspirates to a small volume of EDTA whole blood ( ≤ 0.5mL) to enrich the blood sample (there is no tissue processing fee for this sample). For steps on properly obtaining and submitting lymph node aspirates please see instructions . Other aspirate samples can be submitted on slides. Please make sure to send at least 3-4 slides and make sure they are appropriately labeled with the sample type/source. Slides are usually pooled together for testing to ensure that there is enough sample for DNA extraction.
When sending alternative sample types, it is recommended that the standard EDTA whole blood and serum samples be submitted as well for holding or additional testing.
All types of serum can be submitted for testing. Icterus, hemolysis, and /or lipemia should not interfere with IFA test results.
Any species of animal can be tested by PCR methodology. IFA serology testing, however, can only be performed on canine or feline samples since our assays depend on reagents that are host species specific (see IFA interpretation below). Our first level of PCR testing is performed at a genus level and is usually broad enough to detect vector borne disease pathogens that may infect a wide variety of host species. If a novel/unusual pathogen is detected, the result is verified by additional testing and DNA sequencing. Please reach out to us via phone or email if there are any questions about specific pathogens that you would like to identify in a patient. We are unable to perform diagnostic testing on human samples. If in need of human testing, please contact Galaxy Diagnostics or other diagnostic laboratories approved to test human samples.
Appropriate antimicrobial administration should have minimal to no effect on acute and convalescent serology testing but may decrease the number of circulating organisms to levels below detection by PCR testing. Sample collection prior to antimicrobial treatments is ideal; however, PCR testing may still be useful even after antimicrobial therapy is started. Studies have shown that PCR tests do not immediately become negative after the initiation of antimicrobials. Furthermore, many antibiotics (that are not anti-rickettsial agents) are unlikely to affect vector-borne pathogens. Immunosuppressive therapy is not a contraindication for PCR or serology testing, Use of immunosuppressive therapy may increase the pathogen load in some patients, and its effect on antibody development in vector borne pathogen infections is unclear. Prior immunosuppressive therapy should not deter further vector-borne disease testing.
Immunofluorescent antibody test (IFAT) detects and quantifies antibodies that cross react to whole-cell vector-borne pathogens. IFAT can be performed on animal serum, provided the fluorescent-labeled secondary antibody conjugate is specific for that animal’s antibodies (i.e. dog serum must be tested with canine-specific conjugate and cat serum must be tested with feline-specific conjugate). The VBDDL only performs IFATs on canine and feline serum samples. The VBDDL performs 2-fold serial dilutions (1:2, 1:4, 1:8, 1:16, etc.) of submitted serum samples. Individual diagnostic laboratories may perform different serial dilutions of the serum, and thus may establish different endpoint titers to indicate positive or negative results. Because of this, it is recommended to use the same diagnostic laboratory when comparing IFAT results. The VBDDL considers an end point titer of ≥1:64, positive for exposure.
IFAT is a serological diagnostic modality that can be used to identify exposure to or infection with vector-borne pathogens. Antibodies made in response to pathogens can take several weeks post-exposure to become detectable, thus serology is not appropriate for the diagnosis of most acute diseases. Comparison of antibody titers in serum collected from acute and convalescent time points can indicate an active infection. When comparing acute and convalescent antibody titers, a 4-fold increase (i.e. 2 dilutions if 2-fold serial dilutions were performed on the serum) in the convalescent titer indicates an active infection. Detection of antibodies at a single point in time may represent a past exposure or an active infection.
It is important to remember IFAT depends on a technician for microscopy and visual interpretation of IFAT antigen slides; thus, end-point titer decisions may vary by 1-2 dilutions, depending on visual interpretation difference between different technicians. This is a limitation of IFAT serology.
General Guidelines for Serology Interpretation
- Detects antibodies, not the pathogen so for many vector-borne disease, you may only be identifying a previous exposure or a cross-reaction with a similar organism.
- Not optimal for detecting acute infections (~2 weeks to mount and antibody response).
- A 4 fold or greater increase in titers measured ~4 weeks apart (acute and convalescent samples) indicate a recent active infection (optimal to test samples in parallel) example: 1:64 → 1:256 = 4 fold increase.
- In general, serology is not useful for discrimination of species or subspecies for most vector-borne pathogens.
- Duration of antibodies present after a vector-borne pathogen has been cleared varies.
IFAT is a serological diagnostic modality that detects antibodies against vector-borne pathogens. A negative IFAT result (< 1:64) indicates antibodies were not detected against a specific pathogen. End-point titers of 1:16 and 1:32 are not considered positive due to high degree of non-specific reactivity that can occur when antibodies are highly concentrated. A negative IFAT could indicate the animal was not exposed to a particular vector-borne pathogen or antibodies have not yet been generated in response to infection. Antibodies made in response to pathogens can take several weeks post-exposure to become detectable. False negative results can generate negative titers. If you suspect a false results, please contact the VBDDL and we can perform repeat testing in many instances.
IFAT positive and SNAP®4DX®plus negative
- Titers may be below limit of SNAP4DXplus detection
- New species or strain of pathogen that cross reacts w/ whole cells in IFAT but not peptide in SNAP4DX
- IFA detects earlier Ab response (IgM)
- IFAT false (+) or SNAP®4DX®plus false (-)
IFAT negative and SNAP®4DX®plus positive
- Additional pathogens tested for on SNAP®4DX®plus but not IFAT (e.g. ewingii)
- Dog previously SNAP®4DX®plus (+) (dogs can remain SNAP+ for years)
- IFA false (-)/ SNAP®4DX®plus (+)
SNAP®4DX®plus test kit is designed to detect antibodies seroreactive to tick-borne diseases and uses a conjugate that is not species-specific (e.g. can bind to antibodies from other animals other than dogs); however, it is licensed only for use in dogs. Several studies report using feline and equine sera with SNAP®4DX®plus to detect seroreactivity to Anaplasma spp, Ehrlichia spp, and Borrelia burgdorferi peptides. Clinicians who choose to use serologic tests not licensed for use in a particular animals, should do so with the understanding that the sera may not consistently react with small peptides validated for canine seroreactivity, potentially generating inaccurate results.
A positive PCR test indicates the presence of pathogen DNA in the clinical sample, which supports a recent or current infection. All positive PCRs are confirmed with either DNA sequence analysis and/or a second species-specific PCR. Occasionally we may report results as equivocal if we obtain a weak positive screening result and are unable to repeat or confirm the original positive PCR. In these cases, you will be provided with a free PCR test to use immediately or at a later date for retesting.
PCR is a highly sensitive method for the detection of pathogen DNA; however, false-negative results may occur if pathogen is not in the specimen submitted for testing or if the pathogen load is below the limit of detection. The stage of infection can impact pathogen load. For example, vector-borne pathogen load is typically higher when sampled during the acute phase of an infection. Clinicians should not consider a negative diagnostic PCR result as conclusive evidence for the absence of an infection, but rather that the pathogen DNA was not detected in a particular sample at a particular point in time. Combining molecular and serologic modalities can improve the likelihood of obtaining a diagnosis.
Disease information, treatment recommendations, research publications, and other useful links can be found under the Resources tab listed above
A consultation with one of our clinicians about a vector-borne disease in an animal can be requested at no cost by a veterinarian. Due to the volume of consultation requests to our laboratory we ask that requests only be made by veterinarians submitting samples to our lab for diagnostic testing. We are unable to provide diagnostic consults to owners. A consultation request form can be found under the ‘Consult Request’ tab listed above. Please download and save the form, fill it out, and send it as an attachment via email to email@example.com. From there, a consult will be arranged with one of our three clinical advisors. Consults are generally returned within 48 hours. Please contact our laboratory by phone or email if you have any additional questions.
For all international shippers shipping blood/serum samples from exotic animals (not cats or dogs) please check the CITES appendices (www.Cites.org/eng/app/appendices.php) for a list of species requiring export permits or special certificates.
Additional information about required permits (by country) can be found by reaching out to the contacts on the National CITES authorities list.(https://www.cites.org/cms/index.php/component/cp/)
An import permit is required for most all international shipments. This can be obtained by emailing us at firstname.lastname@example.org. Please make sure to include the species, specimen type, and a copy of your Export Permit or CITES Permit (if available) in the body of your email.
*It is important to note that sending DNA samples instead of blood, serum, or tissue specimens are the preferred for some countries and can be easier to ship
In general, testing can be canceled within 48 hours of receipt of sample if the PCR or Serology testing has not been started yet. All cancellations must be requested in writing via email to email@example.com. Samples from canceled tests will be held for 2 weeks before being discarded.
- CITES (International Shipping of Exotic Specimens) CITES
- How to Avoid Tick Bites
- Intracellular Pathogens Research
- Tick and Vector Testing for Research
- Ticks and Transmitted Diseases in Oklahoma
- Entomology-Image Gallery (Ticks)
- University of Kentucky-Entomology
- CAPC Recommendations
- Galaxy Diagnostics (BAPGM Culture)
- Tick Borne Diseases – On Genome Barks, we welcome back Dr. Ed Breitschwerdt, a specialist in internal medicine and infectious disease at North Carolina State University. Dr. Breitschwerdt has received funding from the AKC Canine Health Foundation for various infectious diseases including Bartonella spp. In this podcast, Dr. Breitschwerdt describes several common tick borne illness, including Rocky Mountain Spotted Fever, Ehrlichiosis and Lyme Disease. Dr. Breitschwerdt also shares the symptoms to watch for and what treatments are available.
- Bartonella: “The Hidden Epidemic!” – On Radio In Vivo. Guest: Dr. Edward Breitschwerdt, Professor of Internal Medicine, NCSU College of Veterinary Medicine; Chief Scientific Officer & Co-founder, Galaxy Diagnostics, Inc. Topics: Detection, diagnosis and treatment of Bartonella infections in animals and humans, vector borne diseases, One Health
Support our Research
This dream is close to being realized through the combined research partnership of Infectious Disease Specialist Dr. Edward Breitschwert and Oncology Genetics Specialist Dr. Matthew Breen. Research is underway at N.C. State, but they need your help. If you (or your client) would like to make a tax-deductible donation (tax deductible) in support of our vector borne disease research, outbreak investigations and pro bono consultations provided by the NC State College of Veterinary Medicine faculty, click the button below.
Your donation could lead patients and their families to a much awaited diagnosis and to receive the gift of health.
Diagnostic services provided under by the Vector Borne Disease Diagnostic Laboratory are completely self-supported by revenue or donations to the Lab. All income derived from diagnostic testing is used to recover costs for reagents, equipment, supplies and for the salaries of the receptionist and technicians who process the specimens submitted for testing.
Over the years, we have supported a spectrum of outreach and educational opportunities using the revenues generated as a result of our diagnostic service offerings. As examples, students at all academic levels and from many national and international locations, have had the experience of working in a diagnostic laboratory or have participated in a focused research project related to vector borne infectious diseases of companion animals or wildlife species. We are proud of the fact that many of our undergraduate and graduate students have subsequently pursued careers in research or medicine (both human and veterinary medicine).
Interactions with veterinarians throughout the world have led to Lab investigations of complicated cases involving single sick animal or disease outbreaks involving entire kennels. The Lab has frequently participated in these case-based or kennel outbreak studies without taking remuneration into consideration. Unfortunately, there is not a CDC or USDA equivalent for companion animal diseases; therefore there is no infrastructure or established source of funds to address unusual illnesses in pets.
Selected Examples of Our Research:
- In response to a referring veterinarian’s call in the mid 1980’s seeking consultation relative to sick and dying Huskies in a newly built kennel in North Carolina, Rocky Mountain Spotted Fever was diagnosed by skin biopsy and first identified as a disease of dogs.
Published article: Canine Rocky Mountain spotted fever: a kennel epizootic. Breitschwerdt EB, Meuten DJ, Walker DH, Levy M, Kennedy K, King M, Curtis B. Am J Vet Res. 1985;46:2124-8
- In the summer of 1997, the Lab undertook an unfunded investigation of a kennel of sick and dying Walker Hounds routinely used for deer hunting in rural North Carolina. A high degree of co-infection was documented by serology and molecular assays. By PCR, of the 27 dogs, 15 were infected with Ehrlichia canis, 9 with E.chaffeensis, 8 with E.ewingii, 3 with Anaplasma phagocytophilum, 9 with A.platys, 20 with a Rickettsia species, 16 with a Bartonella species, and 7 with Babesia canis. Both E. canis and an uncharacterized Rickettsia species appeared to result in chronic or recurrent infection. As an outcome of this study, lessons were learned about the detection of co-infecting species with an appreciation for high risk populations exposed to numerous ticks.
Published article: Kordick SK, Breitschwerdt EB, Hegarty BC, Southwick KL, Colitz CM, Hancock SI, Bradley JM, Rumbough R, Mcpherson JT, MacCormack JN. Coinfection with multiple tick-borne pathogens in a Walker Hound kennel in North Carolina. J Clin Microbiol. 1999 Aug;37(8):2631-8
- Research on dogs from kennels of American pit bull terriers and of racing and retired Greyhounds, established an association between dog breed and Babesia spp. Babesia gibsoni-infected dogs were more likely to be American pit bull terriers and B. canis vogeli infected dogs were more likely to be greyhounds. These studies described the initial detection of a B. gibsoni epidemic amongst the pit bull terrier population.
Published articles:(1) Birkenheuer AJ, Levy MG, Stebbins M, Poore M, Breitschwerdt E. Serosurvey of antiBabesia antibodies in stray dogs and American pit bull terriers and American staffordshire terriers from North Carolina. J Am Anim Hosp Assoc. 2003 Nov-Dec;39(6):551-7. (2) Birkenheuer AJ, Correa MT, Levy MG, Breitschwerdt EB. Geographic distribution of babesiosis among dogs in the United States and association with dog bites: 150 cases (2000-2003). J Am Vet Med Assoc. 2005 Sep 15;227(6):942-7
- Initial description of the demographic and clinical characteristics of feline cytauxzoonosis was obtained through a retrospective study of 34 C.felis infected cats. Pancytopenia and icterus were the most common clinicopathologic abnormalities. Thirty- two cats either died or were euthanatized, and 2 cats survived. Data indicated that veterinarians in the mid-Atlantic region of the USA should consider C felis infection in cats with fever, icterus, and pancytopenia or bicytopenia.
Published article: Birkenheuer AJ, Le JA, Valenzisi AM, Tucker MD, Levy MG, Breitschwerdt EB. Cytauxzoon felis infection in cats in the mid-Atlantic states: 34 cases (1998-2004). J Am Vet Med Assoc. 2006 Feb 15;228(4):568-71
- The initial recognition of Leishmaniasis in the USA came after a veterinarian north of NYC called concerning fatalities amongst some 120 hounds in a breeding and foxhunting kennel. Upon the suspicion of tick borne disease, serological and molecular testing was requested. Extensive workups, mostly unfunded, on all 120 hounds, including cytology and full necropsies on several at NCSU CVM Teaching Hospital finally resulted in a diagnosis of L.infantum. Further epidemiological workups on the part of the CDC defined the outbreak in this kennel and in other foxhound kennels that trained in southern states as an epidemic of Leishmaniasis unrecognized previously in North America. The VBDDL developed specific IFA and PCR assays to address the diagnosis of Leishmaniasis in the future and aided in the screening of all foxhound kennels in the US.
Published article: Gaskin AA, Schantz P, Jackson J, Birkenheuer A, Tomlinson L, Gramiccia M, Levy M, Steurer F, Kollmar E, Hegarty BC, Ahn A, Breitschwerdt EB. Visceral leishmaniasis in a New York foxhound kennel. J Vet Intern Med. 2002 Jan-Feb;16(1):34-44.
CLINICAL STUDY: Equine Vector-Borne Disease (VBD) in horses: This study has ended.
CLINICAL STUDY: Feline Vector-Borne Disease (FVBD) in cats with acute onset fever: This study has ended.
firstname.lastname@example.org | 919.513.8279