Materials and Methods

Infection and blood sample collection—Blood samples were obtained from a company^a that maintains dogs infected with D immitis. Whole blood samples (10 mL of blood/dog, of which 1.25 mL was used for extraction) were collected from 29 D immitis–infected dogs and 10 uninfected dogs in the company's facility at the time of the study. Infected dogs were Beagles that had been previously inoculated with adult or larval D immitis under the guidance of an animal ethics committee. Adult heartworms were inoculated intraventricularly, and larvae were inoculated SC. Prior to blood sample collection, infection in these dogs was confirmed on the basis of results of screening whole blood samples for D immitis antigen. Uninfected dogs were also screened to confirm the absence of circulating D immitis antigen prior to collection of whole blood samples for use in the present study. For 16 of the 29 infected dogs, blood samples were examined for circulating microfilaria at the time of sample collection, and the number of parasites/20 μL was recorded for each dog. Each blood sample was collected in a 7-mL tube containing EDTA, immediately frozen, and transported for analysis.^b

Extraction of DNA from blood samples—A combination of bead-beating cell lysis and magnetic bead isolation was used to extract nucleic acids from whole blood samples obtained from infected and uninfected dogs. For homogenization, 1.25 mL of each whole blood sample was placed in a single 2.0-mL screw-cap tube^c filled with 1.35 g of 0.1-mm yttrium-stabilized zirconium oxide beads.^d To each tube containing 1.25 mL of blood, proteinase K solution^e (25 μL), 20% SDS solution^f (142 μL), extraction control^g (1 μL), and antifoam A^h (10 μL) were added. The mixture was then homogenized in a tissue homogenizerⁱ at 6,200 revolutions/min for 90 seconds 3 times (5-second interval between events); total homogenization time was 270 seconds. Each homogenized lysate was incubated at 56°C for 15 minutes and then centrifuged for 3 minutes at 16,000 × g in a benchtop microcentrifuge. Subsequently, nucleic acids were isolated with a magnetic particle processor.^j One milliliter of lysate was transferred to a 24-well deep-well plate^j along with lysis buffer^g (1.1 mL) and magnetic particles^g (160 μL). Each lysate mixture was incubated for 16.5 minutes in the lysis buffer at 56°C. Specimens were then washed once in wash buffer^k and 3 times in another wash buffer^l (1-minute incubation for each wash step). The magnetic beads were then dried for 3 minutes at 65°C, and nucleic acids were eluted into 250 μL of elution buffer^g by incubating the magnetic particles at 65°C for 3 minutes.

PCR and RT-PCR assay procedures—Detection of heartworm infection was performed with an assay involving PCR primers that were designed to detect a wide range of vector-borne pathogens by targeting conserved regions of DNA that border variable regions (Appendix 1). Primer pairs BCT3511, BCT3517, BCT2328, and INV4855 were run as a multiplex PCR procedure. A 1-step RT-PCR procedure was performed for the reactions containing primer pairs VIR2217 (single-plex reaction) and VIR2230 with BCT3570 (multiplex reaction). The remaining primer pairs were run in singleplex PCR procedures. For one of the primer pairs in the reactions, an internal positive control made from cloned synthetic DNA^m (20 copies/reaction) was included. Each internal control was designed to be identical to the expected amplicon, with the exception of a 5-bp deletion to enable the control to be distinguished from the target-derived amplicon.

The PCR procedure was performed in a 50-μL reaction volume containing 10 μL of nucleic acid extract in a reaction mix that combined a 750nM concentration of each primer as previously described.¹⁰ The 1-step RT-PCR procedure was performed in a 50-μL reaction volume containing 10 μL of nucleic acid extract in a reaction mix that combined a 750nM concentration of each primer as previously described.¹⁴ Because all reactions for a sample were run in the same 96-well RT-PCR plate, cycling conditions were used for both the RT-PCR and PCR procedures as previously described.¹⁴

16S DNA sequence analysis—The presence of the Wolbachia endosymbiont in D immitis–infected dogs was confirmed via 16S DNA sequencing of a Wolbachia-positive specimen with primers 4F and 801R (Appendix 2) to amplify an 800-bp region of the 16S gene. The primers have an M13 tag sequence, which was used for the sequencing reactions. Nucleic acid extract from a blood sample of an infected dog included in the study was used as a representative extract of all D immitis–infected study dogs. Each 16S PCR assay was performed in a 40-μL reaction volume containing 1 μL of nucleic acid extract in a reaction mix composed of 1 U of immolase Taq polymerase, 20mM Tris (pH, 8.3), 75mM KCl, 1.5mM MgCl₂, 0.4M betaine, 200μM deoxyadenosine triphosphate, 200μM deoxycytidine triphosphate, 200μM deoxythymidine triphosphate, 200μM deoxyguanosine triphosphate, 20mM sorbitol, 2 μg of sonicated poly A RNA/mL, 500 μg of ultrapure bovine serum albumin/mL, and a 750nM concentration of each primer. The following PCR cycling conditions were used to generate the 16S amplicons on an 96-well thermocyclerⁿ: 95°C for 10 minutes, followed by 8 cycles of 95°C for 15 seconds, 50°C for 45 seconds, and 72°C for 90 seconds, with the 50°C annealing temperature increasing 0.6°C for each cycle. The PCR assay then continued for 37 additional cycles of 95°C for 15 seconds, 60°C for 15 seconds, and 72°C for 60 seconds. The PCR cycle ended with a final extension of 4 minutes at 72°C, followed by a 4°C hold. The PCR amplicons were sequenced^o with a DNA sequencer.^p

MS and analysis—Mass spectrometry was performed with a biosensor.^q After PCR amplification, 30-μL aliquots of each PCR product were desalted and analyzed via MS as previously described.^10,11,15,16 Dogs were considered infected with D immitis when results yielded the appropriate base count for either the BCT3575 or BCT3570 primer pair.

Results

Infection status of dogs—For all 29 dogs that had been inoculated with D immitis (adult or larval forms), circulating D immitis antigen was detected in the blood samples at the time of collection. Samples were collected from the infected dogs at 3 to 55 months after inoculation (Table 1). Blood samples from all uninfected dogs yielded negative results for circulating D immitis antigen.

Detection of D immitis infections in canine blood samples via the PCR–ESI-MS assay—Whole blood samples from 29 dogs infected with D immitis and 10 uninfected dogs were used to estimate the ability of the assay under investigation (which was designed to detect a wide range of clinically relevant vector-borne pathogens) to identify D immitis infection in dogs via detection of the obligate Wolbachia endosymbiont of D immitis. Two of the assay primer pairs, BCT3575 and BCT3570, target Alphaproteobacteria and result in specific amplicons in the blood samples from dogs infected with D immitis (Figure 1). These signatures were not observed in blood samples collected from uninfected dogs. These base count signatures were also unique, compared with those of other Alphaproteobacteria that have been screened with these primers (data not shown).

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Results of a PCR–ESI-MS assay performed with each of 2 primer pairs (BCT3575 [A] and BCT3570 [B]) to detect the Wolbachia endosymbiont of Dirofilaria immitis in a whole blood sample collected from a dog in which heartworm infection had been experimentally induced. For Wolbachia endosymbiont amplicon detection, primer pair BCT3575 targets rpoB and primer pair BCT3570 targets gltA; targets are conserved regions of endosymbiont DNA that border variable regions. The mass for both the forward and reverse strands of the amplicons are given. Between the major peaks in each panel, the numbers after the letters A, G, C, and T represent the amplicon base composition (ie, the number of adenine [A], guanine [G], cytosine [C], and thymine [T] bases).

Citation: American Journal of Veterinary Research 73, 6; 10.2460/ajvr.73.6.854

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Blood samples collected from 16 of the 29 infected dogs were examined for circulating microfilaria at the time of sample collection; microfilarial status was not determined for the other 13 infected dogs. In the 13 D immitis antigen–positive blood samples for which the microfilarial status was unknown, the Wolbachia endosymbiont of D immitis was detected by BCT3575 and BCT3570 in all samples and in 9 samples, respectively (Table 1). In 11 blood samples in which microfilaria concentrations ranged from 2 to 460 microfilaria/20 μL of blood, the Wolbachia endosymbiont of D immitis was detected by BCT3575 and BCT3570 in all samples and in 10 samples, respectively. Two dogs had occult infections; the blood samples collected from those dogs were D immitis–antigen positive but microfilaria negative. The endosymbiont was not detected in the blood samples collected from dogs with occult heartworm infections. Additionally, the endosymbiont was not detected with either primer pair in blood samples collected from 3 dogs that had been inoculated with D immitis larvae 3 months earlier. The Wolbachia endosymbiont of D immitis was not detected by BCT3575 or BCT3570 in any blood sample collected from the 10 uninfected dogs. For all blood samples (from infected and uninfected dogs), no other pathogens were detected via the PCR–ESI-MS assay.

Identification of the Wolbachia endosymbiont of D immitis via 16S DNA sequencing—The presence of the Wolbachia endosymbiont of D immitis in the blood samples collected from infected dogs was confirmed via sequencing of the 16S amplicon from a representative sample of 1 infected dog. The sequencing results obtained matched that of the previously sequenced Wolbachia endosymbiont of D immitis (719/720 nucleotides; GenBank accession No. AF088187). No other 16S sequence was detected in the blood sample collected from that D immitis–infected dog.

Discussion

Companion and domestic animals can become infected with a wide range of vector-borne pathogens (bacteria, viruses, protozoa, and nematodes), which can be both life-threatening and difficult to diagnose. These animals also can serve as sentinel species for the spread of vector-borne pathogens to humans.^17,18 To this end, we have developed a single broad-range PCR–ESI-MS assay to detect and identify a wide range of vector-borne pathogens important to animal health, including Spirochaetes (eg, Borrelia and Leptospira spp), Gammaproteobacteria (eg, Francisella spp), Alphaproteobacteria (eg, Ehrlichia, Anaplasma, Rickettsia, Bartonella, and Wolbachia spp), flaviviruses (eg, Powassan virus and tick-borne encephalitis virus), and protozoa (eg, Babesia spp). In the present study, we demonstrated the ability of this assay to identify D immitis infection in dogs via detection of the heartworm Wolbachia endosymbiont in whole blood samples. The entire assay process, from specimen preparation to result reporting, can be completed within 6 hours for 1 sample; moreover, the biosensor system^q used in the present study has a potential throughput of > 150 samples/24 h. Assay results provide veterinarians with useful information about a wide range of infectious organisms, including coinfections. To our knowledge, this is the first report of heartworm infection in dogs that was directly detected via assessment of blood samples by use of the PCR–ESI-MS assay.

Identification of D immitis infection has been routinely performed via microscopic detection of the circulating microfilaria or detection of circulating D immitis antigen via an ELISA or other immunochromatographic tests. Visual detection of circulating microfilaria requires examination of ≤ 1 mL of blood by a skilled technician, and a positive result can be overlooked. It has been previously reported that even in areas with a high prevalence of heartworm infection, approximately 20% of the infected dogs may not have circulating microfilaria, which creates challenges for visual detection methods.¹⁹ In the present study, the Wolbachia endosymbiont was detected in all blood samples that contained microfilaria, but it was not detected in blood samples from 2 dogs with occult infections, suggesting that circulating microfilaria might be required to detect the heartworm endosymbiont in canine blood samples. However, further controlled investigations of blood samples collected from dogs with occult infections would be needed to test this hypothesis. Nevertheless, results of the present study have suggested that the PCR–ESI-MS assay could be used in place of microscopy to detect D immitis infections in dogs with circulating microfilaria.

Similar to D immitis, Wolbachia bacteria are harbored by many other filarial nematodes, including pathogens of other domesticated animals (eg, Parafilaria bovicola in horses and Onchocerca spp in cattle) and humans (eg, W bancrofti, Onchocerca volvulus, and Brugia malayi).⁶ Due to the broad-range design of the assay, it may be possible to detect these and other filarial nematode infections in humans and other animals via detection of their respective obligate endosymbionts, although further studies would be required.

Heartworm is one of many vector-borne diseases that infect dogs. In the study reported here, a broad-range PCR–ESI-MS assay designed to detect vector-borne pathogens was used to identify D immitis infection in dogs through detection of the heartworm Wolbachia endosymbiont in whole blood samples. By use of the assay, whole blood samples collected from experimentally infected dogs with circulating microfilaria were analyzed and results confirmed D immitis infection. This assay can also detect a wide range of vector-borne bacterial, viral, and protozoal pathogens (including co-infections) in a single blood sample. Further controlled studies to evaluate the use of this PCR–ESI-MS assay for the diagnosis of D immitis infection in dogs in clinical settings are warranted.