DOI: 10.5061/dryad.ttdz08ktd
title: Molecular prevalence of Bartonella, Babesia, and hemotropic Mycoplasma species in dogs with hemangiosarcoma from across the United States

description: Hemangiosarcoma (HSA), a locally invasive and highly metastatic
 endothelial cell neoplasm, accounts for two-thirds of all cardiac and
 splenic neoplasms in dogs. Bartonella spp. infection has been reported in
 association with neoplastic and non-neoplastic vasoproliferative lesions
 in animals and humans. The objective of this study was to determine the
 prevalence of Bartonella spp. in conjunction with two other hemotropic
 pathogens, Babesia spp. and hemotropic Mycoplasma spp., in tissues and
 blood samples from 110 dogs with histopathologically diagnosed HSA from
 throughout the United States. This was a retrospective, observational
 study using clinical specimens from 110 dogs with HSA banked by the
 biospecimen repository of the Canine Comparative Oncology and Genomics
 Consortium. Samples provided for this study from each dog included:fresh
 frozen HSA tumor tissue (available from n = 100 of the 110 dogs), fresh
 frozen non-tumor tissue (n = 104), and whole blood and serum samples (n =
 108 and 107 respectively). . Blood and tissues were tested by qPCR for
 Bartonella, hemotropic Mycoplasma, and Babesia spp. DNA; serum was tested
 for Bartonella spp. antibodies. Bartonella spp. DNA was amplified and
 sequenced from 73% of dogs with HSA (80/110). In contrast, hemotropic
 Mycoplasma spp. DNA was amplified from a significantly smaller proportion
 (5%, p&lt;0.0001) and Babesia spp. DNA was not amplified from any dog.
 Of the 100 HSA tumor samples submitted, 34% were Bartonella PCR positive
 (32% of splenic tumors, 57% of cardiac tumors, and 17% of other tumor
 locations). Of 104 non-tumor tissues, 63% were Bartonella PCR positive
 (56% of spleen samples, 93% of cardiac samples, and 63% of
 skin/subcutaneous samples). Of dogs with Bartonella positive HSA tumor,
 76% were also positive in non-tumor tissue. Bartonella spp. DNA was not
 PCR amplified from whole blood. This study documented a high prevalence of
 Bartonella spp. DNA in dogs with HSA from geographically diverse regions
 of the United States. While 73% of all tissue samples from these dogs were
 PCR positive for Bartonella DNA, none of the blood samples were,
 indicating that whole blood samples do not reflect tissue presence of this
 pathogen. Future studies are needed to further investigate the role of
 Bartonella spp. in the development of HSA.
description: <b>Study design and sample sources </b>
 This was a retrospective, observational, descriptive study of 110
 dogs with HSA. Specimens used for this study were previously collected and
 banked by the Canine Comparative Oncology and Genomics Consortium (CCOGC)
 based on previously published standard operating procedures.[34]. Briefly,
 the CCOGC collected samples from eight participating veterinary university
 teaching hospitals starting in 2006 with the goal of creating a repository
 of clinical samples from dogs with common naturally occurring cancers.
 Prior to sample submission to CCOGC, dogs were given a definitive
 diagnosis of neoplasia based on histopathology performed by a
 board-certified veterinary pathologist at the diagnostic laboratory of
 each participating university. Tissue samples for the CCOGC biospecimen
 repository were obtained from the primary tumor via surgical biopsy or
 post-mortem collection (HSA tumor tissue). As an internal control, tissue
 samples from each dog were also obtained from adjacent grossly normal
 tissue in the same organ, or if no grossly normal tissue in the affected
 organ was apparent, skin biopsies were obtained (non-tumor tissue). Tissue
 submission (HSA tumor and non-tumor) from each dog required provision of
 both formalin-fixed (subsequently stored in ethanol) and non-fixed
 specimens snap-frozen in liquid nitrogen (subsequently stored at -80° C).
 Dogs also had serum and whole blood collected at the time of tissue
 sampling for submission to the CCOGC. For this study,
 samples from dogs diagnosed with HSA were provided by the CCOGC repository
 to the investigators. Four sample types were provided for pathogen
 testing: fresh frozen HSA tumor tissue, fresh frozen non-tumor tissue,
 whole blood, and serum. Two sample types were provided for
 histopathological confirmation of tissue type and tumor presence included:
 formalin-fixed HSA tumor tissue and formalin-fixed non-tumor tissue. Any
 dog that had a diagnosis of HSA (as determined by the CCOGC SOP), and had
 an adequate amount of fresh frozen tissue stored in the biorepository at
 the time of the investigators’ request for specimens, was included. This
 study, therefore, included 110 dogs with samples collected between May
 2008 and November 2011. Because of previous sample
 requests or lack of submission of all requested samples to the CCOGC,
 there were samples missing when provided to the investigators. Of the 110
 dogs, 91 had a complete set of the 4 sample types for pathogen testing
 (fresh frozen HSA tumor tissue, fresh frozen non-tumor tissue, whole
 blood, serum) provided to the investigators. No dog was missing more than
 one sample type, and all dogs had at least one fresh-frozen tissue sample
 (HSA tumor, non-tumor tissue, or both) available for testing. For this
 reason, dogs with missing samples for pathogen testing were not excluded
 from the study. Similarly, of the 110 dogs only 37 had a complete set of
 the 2 sample types for histopathological confirmation (formalin-fixed HSA
 tumor tissue, formalin-fixed non-tumor tissue) provided to the
 investigators. Of the 110 dogs, 37 had formalin-fixed HSA tumor tissue and
 93 had formalin-fixed non-tumor tissue provided. There were no
 formalin-fixed samples of HSA tumor or non-tumor tissue from cardiac
 tumors available for independent histopathologic review. Because a large
 proportion of dogs were missing formalin-fixed samples for independent
 histopathological confirmation, the subgroups of tissues with independent
 histopathologic confirmation of tissue type and tumor presence was
 analyzed separately. The CCOGC provided demographic
 information for each dog, including age (years), breed, weight (kg) and
 sex and neuter status. The date and geographic location (university
 teaching hospital) of sample collection was also provided. The anatomic
 location of tumor and non-tumor tissue samples for each dog was also
 provided. <b>Independent confirmation of
 demographic and histologic data</b> When
 possible, information provided by the CCOGC was independently confirmed by
 the investigators for this study. Histopathology reports providing the
 diagnosis of hemangiosarcoma wwere provided for 102 dogs; these were
 reviewed by one author (EL) to confirm hemangiosarcoma diagnosis and tumor
 location. For dogs with formalin-fixed biopsy samples
 provided (see above), biopsies were independently evaluated by a
 board-certified veterinary pathologist (KL) to confirm the tissue and
 tumor origin of the biopsy. Formalin fixed tissues were submitted to the
 NCSU College of Veterinary Medicine histology laboratory (Raleigh NC) and
 tissues were embedded in paraffin blocks (FFPE blocks). Slides containing
 5 um sections were prepared from the FFPE blocks and stained with
 hematoxylin and eosin (H&E). Samples were categorized by organ
 type and HSA tumor or non-tumor tissue based on H&E staining. If
 the tissue of origin was not able to be determined from the biopsy (5
 tumor samples and 2 non-tumor tissues), or if no formalin-fixed sample was
 provided (68 tumor samples and 14 non-tumor tissues), the tissue of origin
 was categorized by the information provided by the CCOGC and the original
 diagnostic histopathology reports. Non-tumor tissues from skin or various
 subcutaneous tissues (including hair, skin, adipose, skeletal muscle, or
 mammary gland, or any combination of these tissues) were categorized as
 skin/SQ for analysis. <b>Pathogen detection
 methods</b> DNA was extracted from EDTA
 anti-coagulated blood and fresh frozen tissue samples using a Qiagen
 DNeasy® Blood and Tissue kit (Qiagen, Valencia, CA) following the
 manufacturer’s protocols. For each tissue sample (HSA tumor and
 non-tumor), a 25 mg piece of tissue was excised from the entire sample
 using a new, prepackaged sterile scalpel. DNA yield and quality was
 assessed by spectrophotometry (Nanodrop, Wilmington, DE).
 Each DNA sample was screened for the presence of
 <i>Bartonella</i> spp. DNA using conventional and qPCR, and
 <i>Babesia </i>spp. and hemotropic
 <i>Mycoplasma</i> spp. using qPCR. <i>Bartonella
 </i>qPCR was performed using primers targeting the 16S-23S
 intragenic transcribed spacer (ITS) region of <i>Bartonella
 </i>species as described previously[35], in conjunction with a
 BsppITS438 FAM-labeled
 hydrolysis probe (TaqMan, Applied Biosystems, Foster City,
 CA, USA).
 <i>Bartonella </i>qPCR was performed at two dilutions for each
 sample (using 1 uL and 5 uL of template DNA respectively). PCR screening
 for <i>Babesia</i> spp. and hemotropic <i>Mycoplasma
 </i>spp. were carried out as described
 previously.[22] Briefly, oligonucleotides
 Myco16S-322s (5’ GCCCATATTCCTACGGGAAGCAGCAGT 3’) and Myco16S-938as (5’
 CTCCACCACTTGTTCAGGTCCCCGTC 3’) were used as forward and reverse primers
 respectively for hemotropic <i>Mycoplasma </i>spp. DNA
 amplification. Oligonucleotides Piro18S-144s (5’
 GATAACCGTGSTAATTSTAGGGCTAATACATG 3’) and Piroplasma18S-722as (5’
 GAATGCCCCCAACCGTTCCTATTAAC 3’) were used as forward and reverse primers
 respectively for <i>Babesia </i>spp. DNA
 amplification. Amplification was performed in a
 25-µl final volume reaction containing 12.5 µl of MyTaq Premix (Bioline),
 0.2 µl of 100 µM of each forward primer, reverse primer (IDT® DNA
 Technology), 7.1 µl of molecular–grade water, and 5 µl of DNA from each
 sample tested.  PCR negative controls were prepared using 5 µl of DNA from
 blood of a healthy dog. Positive controls for PCR were prepared by using 5
 µl of DNA from previously characterized positive dog (clinical cases). 
 Conventional PCR was performed in an Eppendorf Mastercycler EPgradient®
 under the following conditions: a single hot-start cycle at 95°C for 2
 minutes followed by 55 cycles of denaturing at 94°C for 15 seconds,
 annealing at 68°C for 15 seconds, and extension at 72°C for 18 seconds. 
 Amplification was completed by an additional cycle at 72°C for 1 minute,
 and products were analyzed by 2% agarose gel electrophoresis with
 detection using ethidium bromide under ultraviolet
 light. Validation of positive results was
 performed by Sanger sequencing of amplicons followed by chromatogram
 evaluation and sequence alignment using Contig-Express and Align X
 software (Vector NTI Suite 10.1, Invitrogen Corp, CA, USA). For bacterial
 species identification, DNA sequences were analyzed for nucleotide
 sequence homology at NCBI nucleotide database using BLAST version 2.0. A
 sample was considered <i>Bartonella </i>spp. PCR positive if
 one or more PCR tests (qPCR or conventional PCR) were positive (tests run
 in parallel). Stringent processing methods were used to avoid DNA
 carryover during tissue processing.[36] Specifically, tissue samples were
 processed independently using manual DNA extraction. For all batches of
 DNA extractions, between 2 and 4 blanks samples (water) were used as
 negative controls.  All negative controls for DNA extractions rendered
 negative results on all PCR assays. DNA carryover after PCR amplification
 was avoided by processing each sample in three separate laboratory rooms
 (one for sample sorting and DNA extraction, a second for PCR processing,
 and a third for PCR analysis post amplification), and strict use of
 personal protective equipment for sample handling by laboratory
 personnel. For IFA testing,
 <i>Bartonella</i> antibodies were determined using 3 cell
 culture grown <i>Bartonella </i>spp. (<i>Bartonella
 henselae</i>, <i>Bartonella vinsonii </i>subsp.
 <i>berkhoffii</i>, and <i>Bartonella
 koehlerae</i>) as antigens and following standard immunofluorescent
 antibody assay (IFA) techniques.[37,38] Briefly, bacterial colony isolates
 were passed from agar plate grown cultures into permissive cell lines. 
 For each antigen, heavily infected cell cultures were spotted onto 30-well
 Teflon-coated slides (Cel-Line/Thermo Scientific), air-dried,
 acetone-fixed, and stored frozen. Fluorescein conjugated goat anti-dog IgG
 (KPL, SeraCare, Milford MA) was used to detect bacteria within cells using
 a fluorescent microscope (Carl Zeiss Microscopy, LLC, Thornwood NY). Serum
 samples were diluted in phosphate-buffered saline (PBS) solution
 containing normal goat serum, Tween-20, and powdered nonfat dry milk to
 block nonspecific antigen binding sites. Sera were first screened at
 dilutions of 1:16 to 1:64. All sera that are reactive at 1:64 were further
 tested with two-fold dilutions to 1:8192.
description: Dog_ID: A unique anonymized identifier for each dog
 Collection_Site: The veterinary hospital at which the dog was
 enrolled CensusRegion: Classification of the
 Collection_Site into a census region as follows. Pacific, Pac (WA, OR, CA); Mountain (ID, NV, MT,
 WY, UT, CO, AZ, NM); West North Central, WNC (ND, SD, NE, KS, MN, IA, MO);
 West South Central, WSC (OK, AR, TX, LA); East North Central, ENC (WI, IL,
 IN, OH, MI); East South Central, ESC (KY, TN, MS, AL); South Atlantic (MD,
 DE, WV, VA, NC, SC, GA, FL, DC); Middle Atlantic, MA (NY, PA, NJ); New
 England, NE (ME, NH, VT, MA, CT)
 Collection_Date: Date dog was enrolled
 Collection_Season: Classification of the Collection_Date into a
 season as follows. summer (Jun, July, Aug), autumn (Sept, Oct, Nov),
 winter (Dec, Jan, Feb), spring (Mar, Apr, May) Breed:
 Dog breed reported by veterinary hospital Age_yr: Dog
 age in years, rounded to integer Sex: Dog sex and
 neuter status as follows. Male intact, MI; Male castrated, MC; Female
 intact, FI; Female spayed, FS Weight_kg: Dog weight in
 kilograms Tumor_Organ: The anatomic location of the HSA
 tumor submitted to CCOGC. "EXCLUDE" indicates the tumor tissue
 submitted was not HSA tumor based on independent pathology
 review. NonTumor_Organ: The anatomic location of the
 non-tumor tissue submitted to CCOGC. "EXCLUDE" indicates the
 tumor tissue submitted was not HSA tumor based on independent pathology
 review. "MISSING" indicates tissue was not available from
 CCOGC. Bart_IFA: Bartonella IFA result. A dog was
 considered positive if seroreactivity was 1:64 or greater to one or
 more <em>Bartonella </em>spp. antigen. 0 = negative, 1 =
 positive. "MISSING" indicates serum was not available from
 CCOGC. Any_Sample_Bart_PCR: Bartonella PCR result in
 any sample. A dog was considered positive if PCR was positive on any one
 or more sample (HSA tumor, non-tumor tissue, blood). 0 = negative, 1 =
 positive Blood_Bart_PCR: Bartonella PCR result on blood
 sample. 0 = negative, 1 = positive. "MISSING" indicates blood
 was not available from CCOGC. NonTumor_Bart_PCR:
 Bartonella PCR result on non-tumor tissue. 0 = negative, 1 =
 positive. EXCLUDE" indicates the tumor tissue submitted was not HSA
 tumor based on independent pathology review. "MISSING" indicates
 tissue was not available from CCOGC.
 Tumor_Bart_PCR: Bartonella PCR result on HSA tumor tissue. 0 =
 negative, 1 = positive. "EXCLUDE" indicates the tumor tissue
 submitted was not HSA tumor based on independent pathology
 review. Blood_Myco_PCR: Hemotropic Mycoplasma spp. PCR
 result on blood sample. 0 = negative, 1 = positive. "MISSING"
 indicates blood was not available from CCOGC.
 NonTumor_Myco_PCR: Hemotropic Mycoplasma spp. PCR result on
 non-tumor tissue. 0 = negative, 1 = positive. EXCLUDE" indicates the
 tumor tissue submitted was not HSA tumor based on independent pathology
 review. "MISSING" indicates tissue was not available from
 CCOGC. Tumor_Myco_PCR: Hemotropic Mycoplasma spp. PCR
 result on HSA tumor tissue. 0 = negative, 1 =
 positive. "EXCLUDE" indicates the tumor tissue submitted was not
 HSA tumor based on independent pathology review.
 Blood_Bab_PCR: Babesia spp. PCR result on blood sample. 0 =
 negative, 1 = positive. "MISSING" indicates blood was not
 available from CCOGC. NonTumor_Bab_PCR: Babesia spp.
 PCR result on non-tumor tissue. 0 = negative, 1 = positive. EXCLUDE"
 indicates the tumor tissue submitted was not HSA tumor based on
 independent pathology review. "MISSING" indicates tissue was not
 available from CCOGC. Tumor_Bab_PCR: Babesia spp. PCR
 result on HSA tumor tissue. 0 = negative, 1 =
 positive. "EXCLUDE" indicates the tumor tissue submitted was not
 HSA tumor based on independent pathology review.
 Tumor_HE_Organ: Anatomic origin of HSA tumor tissue, based on
 independent evaluation of formalin-fixed tissues by a single investigator
 (board-certified veterinary pathologist (KL)). "MISSING"
 indicates formalin-fixed tissue was not available from CCOGC.
 Tumor_HE_Path: Pathology in HSA tumor tissue, based on
 independent evaluation of formalin-fixed tissues by a single investigator
 (board-certified veterinary pathologist (KL)). Pathology was confirmed as
 HSA (HSA, HSA equivocal); if pathology was not able to be confirmed
 (Undetermined) the sample was excluded from analysis. "MISSING"
 indicates formalin-fixed tissue was not available from CCOGC.
 NonTumor_HE_Organ: Anatomic origin of non-tumor tissue, based on
 independent evaluation of formalin-fixed tissues by a single investigator
 (board-certified veterinary pathologist (KL)). "MISSING"
 indicates formalin-fixed tissue was not available from CCOGC.
 NonTumor_HE_Path: Pathology in non-tumor tissue, based on
 independent evaluation of formalin-fixed tissues by a single investigator
 (board-certified veterinary pathologist (KL)). Lack of pathology was
 confirmed (Normal); if tumor pathology was seen (HSA) the sample was
 included in the HSA tumor tissue group. If there was pathology that was
 not HSA (Undetermined), the sample was included in the non-tumor tissue
 group. "MISSING" indicates formalin-fixed tissue was not
 available from CCOGC.