Vector Borne Disease

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 dccrowel@ncsu.edu

For specific sample requirements per test, please see our full test catalog above.

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 (we do not recommend PCR testing on separated serum due to low cellularity in the samples, but it can be done). An additional charge of $75 must be added to cover the costs of the special handling required to extract DNA manually 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). 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.

Samples that are drawn should be stored in the refrigerator for up to 2 weeks prior to testing. We do NOT recommend freezing samples, as the freeze/thaw process can affect the DNA structure, and therefore PCR results.

If the patient has already begun treatment and the samples have been in the refrigerator for over 2 weeks, then we still recommend testing the pre-treatment sample anyway.

All samples are stored for at least 2 years. Samples can be removed from long-term storage in the event that additional testing is needed during this time period. Add-on testing availability is dependent upon the sample volume requirements for each test and the amount of sample available in long-term storage. As a veterinary research laboratory we also reserve the right to use archived samples for research purposes, always respecting privacy rights of the contributing animal, owner and veterinarian.

Samples can be shipped overnight via UPS or FedEX to:

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.

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.

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 ncstatevectorborne@ncsu.edu. 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

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.

All test cancellations must be submitted in writing via email to ncstatevectorborne@ncsu.edu within 24 business hours of the sample being received in our laboratory. Testing that has already been completed or begun is unable to be canceled, and we will confirm with you via email what testing (if any) will be invoiced.

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 interpreting serology testing:

1. 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.
2. Not optimal for detecting acute infections (~2 weeks to mount and antibody response).
3. A 4 fold or greater increase in titers measured ~2-4 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.
4. In general, serology is not useful for discrimination of species or subspecies for most vector-borne pathogens.
5. 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.

• Even the most sensitive DNA detection methods can produce false negatives due to extremely low levels of DNA in a given sample. We overcome this testing limitation by enriching samples for three weeks in a patented enrichment media called BAPGM (Bartonella Alpha Proteobacteria Growth Medium), which increases the bacterial load in patient samples up to detectable levels for PCR testing.
• We run PCRs both before and after enrichment culture to ensure that we capture any viable or nonviable DNA present in the sample.

We take special care in making sure that our testing is thorough and results are reported accurately. Our PCR and Serology testing takes about 10 business days to complete from the date that the sample is received in our lab. Our turn-around time can also vary in cases where results need to be confirmed or repeated.

Our BAPGM testing can take up to 30 days to complete. PCR testing is performed on day 0, 7, 14, and 21 of the culture, so preliminary reports will be delivered as those results are finalized.

Please contact us by email at ncstatevectorborne@ncsu.edu with any preliminary result inquiries.

Our lab system accessions testing by test type. You will receive a separate report for each test (SNAP4DX, Serology, PCR, etc.). These reports will say “FINAL” at the bottom of each one. Results are sent out automatically via email by our laboratory system as they are finalized. Once the final results are put in, you will receive a full final report.

Our lab system is only able to have one email address listed per clinic. We strongly recommend that this email address is a shared hospital address rather than a particular doctor or staff member. We also recommend adding ncstatediagnosticlaboratories@ncsu.edu to your email contacts. This way emails are not automatically filtered into the SPAM or JUNK folders.

Our lab system requires a Veterinarian for every submission. If the veterinarian is left blank on the form, or if the full first and last name of the veterinarian is not provided, we will select a default veterinarian from the list to use instead. Please make sure that the first and last name are provided for every submission to avoid this issue.

Our clinicians are available to consult on cases with veterinarians. To request a consult, please send us an email at ncstatevectorborne@ncsu.edu to request our online consult form. Our clinicians are not available to consult directly with owners regarding their pets.

If you are unable to access the online consult form, please reach out to us via email at ncstatevectorborne@ncsu.edu and let us know that you are unable to access the form.

Our clinicians do their best to reach out as soon as they are available. The typical timeframe to expect a response is within 2 business days. If you have not heard back from a clinician within this time frame, please send us an email at ncstatevectorborne@ncsu.edu so that we can touch base with the clinicians and someone can reach out to discuss the case.

The VBDDL is open Monday through Friday from 9am EST until 5pm EST.

Email is the best form of contact for us here at the VBDDL. You can also call and leave us a voicemail. We try to return calls and emails within 24 business hours.

We recommend going to www.tickcheck.com for testing and identifying ticks after removal.

We recommend the Companion Animal Parasite Council for information regarding vector borne diseases in animals.

We recommend the CDC Website for information regarding vector borne diseases in humans.

1. Rickettsia typhi infection in a clinically-ill dog from Houston, Texas; https://www.sciencedirect.com/science/article/pii/S2405939022000971
2. Detection of Vector-Borne Infections in Lions and Tigers at Two Zoos in Tennessee and Oklahoma, USA;
   https://bioone.org/journals/journal-of-zoo-and-wildlife-medicine/volume-53/issue-1/2020-0199/DETECTION-OF-VECTOR-BORNE-INFECTIONS-IN-LIONS-AND-TIGERS-AT/10.1638/2020-0199.short
3. Visceral leishmaniasis in a New York foxhound kennel https://onlinelibrary.wiley.com/doi/pdf/10.1111/j.1939-1676.2002.tb01604.x
4. Serosurvey of anti-Babesia antibodies in stray dogs and American pit bull terriers and American staffordshire terriers from North Carolina https://www.researchgate.net/publication/8908189_Serosurvey_of_Anti_Babesia_Antibodies_in_Stray_Dogs_and_American_Pit_Bull_Terriers_and_American_Staffordshire_Terriers_From_North_Carolina
5. Efficacy of combined atovaquone and azithromycin for therapy of chronic Babesia gibsoni (Asian genotype) infections in dogs. https://onlinelibrary.wiley.com/doi/pdf/10.1111/j.1939-1676.2004.tb02573.x
6. Efficacy of Atovaquone and Azithromycin or Imidocarb Dipropionate in Cats with Acute Cytauxzoonosis https://onlinelibrary.wiley.com/doi/full/10.1111/j.1939-1676.2010.0646.x
7. Re-emergence of Babesia conradae and effective treatment of infected dogs with atovaquone and azithromycin https://www.sciencedirect.com/science/article/pii/S030440171200009X
8. Infection with Panola Mountain Ehrlichia sp. in a dog with atypical lymphocytes and clonal T-Cell expansion https://onlinelibrary.wiley.com/doi/full/10.1111/jvim.12148
9. Regional Seroreactivity and Vector-Borne Disease Co-Exposures in Dogs in the United States from 2004–2010: Utility of Canine Surveillance https://www.liebertpub.com/doi/full/10.1089/vbz.2014.1592
10. A serological survey of tick-borne pathogens in dogs in North America and the Caribbean as assessed by Anaplasma phagocytophilum, A. platys, Ehrlichia canis, E. chaffeensis, E. ewingii, and Borrelia burgdorferi species-specific peptides  https://www.tandfonline.com/doi/full/10.3402/iee.v4.24699
11. Serological and molecular analysis of feline vector-borne anaplasmosis and ehrlichiosis using species-specific peptides and PCR https://link.springer.com/article/10.1186/s13071-015-0929-8
12. Improved molecular detection of Babesia infections in animals using a novel quantitative real-time PCR diagnostic assay targeting mitochondrial DNA https://link.springer.com/article/10.1186/s13071-017-2064-1
13. Bartonella Seroepidemiology in Dogs from North America, 2008–2014 https://onlinelibrary.wiley.com/doi/full/10.1111/jvim.14890
14. Clinical, morphological, and molecular characterization of an undetermined Babesia species in a maned wolf (Chrysocyon brachyurus) https://www.sciencedirect.com/science/article/pii/S1877959X18300463
15. Babesia gibsoni cytochrome b mutations in canine blood samples submitted to a US veterinary diagnostic laboratory https://onlinelibrary.wiley.com/doi/full/10.1111/jvim.15300
16. Genetic conservation of Cytauxzoon felis antigens and mRNA expression in the schizont life-stage https://www.sciencedirect.com/science/article/pii/S0304401718303443
17. Clinicopathological findings in 41 dogs (2008‐2018) naturally infected with Ehrlichia ewingii https://onlinelibrary.wiley.com/doi/full/10.1111/jvim.15354
18. An update on the treatment of canine monocytic ehrlichiosis (Ehrlichia canis) https://www.sciencedirect.com/science/article/pii/S1090023318306488
19. Feline Vector-Borne Diseases in North America https://www.vetsmall.theclinics.com/article/S0195-5616(19)30042-7/abstract
20. Prevalence of Babesia spp. and clinical characteristics of Babesia vulpes infections in North American dogs https://onlinelibrary.wiley.com/doi/full/10.1111/jvim.15560
21. Ecological and Socioeconomic Factors Associated with Bartonella henselae Exposure in Dogs Tested for Vector-Borne Diseases in North Carolina https://www.liebertpub.com/doi/full/10.1089/vbz.2018.2397
22. Molecular identification of vector-borne organisms in Ehrlichia seropositive Nicaraguan horses and first report of Rickettsia felis infection in the horse https://www.sciencedirect.com/science/article/pii/S0001706X1930782X
23. Babesia sp. infection in a zoo-housed polar bear (Ursus maritimus) https://www.sciencedirect.com/science/article/abs/pii/S2405939019302618#:~:text=A%2028%2Dyear%2Dold%20female,the%20bear%20was%20humanely%20euthanized.
24. A retrospective study of vector‐borne disease prevalence in dogs with proteinuria: Southeastern United States https://onlinelibrary.wiley.com/doi/full/10.1111/jvim.15610
25. Molecular prevalence of Bartonella, Babesia, and hemotropic Mycoplasma species in dogs with hemangiosarcoma from across the United States https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0227234
26. Bartonella rochalimae, a newly recognized pathogen in dogs https://onlinelibrary.wiley.com/doi/full/10.1111/jvim.15793
27. Comparison of Anaplasma and Ehrlichia species – specific peptide ELISAs with whole organism-based immunofluorescent assays for serologic diagnosis of anaplasmosis and ehrlichiosis in dogs
28. Novel Rickettsia Species Infecting Dogs, United States
29. Molecular Characteristics of Rickettsia in Ticks Collected along the Southern Border of Mongolia
30. Demographics and travel history of imported and autochthonous cases of leishmaniosis in dogs in the United States and Canada, 2006 to 2019

Accreditation

• CITES (International Shipping of Exotic Specimens)
• CDC – How to Avoid Tick Bites
• NCSU Intracellular Pathogens Research Laboratory
• Entomology-Image Gallery (Ticks)
• Companion Animal Parasite Council Recommendations

• Babesia
• Bartonella
• Ehrlichiosis & Anaplasmosis
• Leishmaniasis
• Lyme Disease
• Cytauxzoonosis
• Hepatozoonosis