Tabar et al. Parasites & Vectors (2022) 15:105 https://doi.org/10.1186/s13071-022-05205-x Parasites & Vectors RESEARCH Open Access Detection of vector‑borne pathogens in owned dogs with cranial cruciate ligament rupture living in the Mediterranean area María‑Dolores Tabar1, Javier Tabar1, Carolina Naranjo2, Laura Altet3 and Xavier Roura4* Abstract Background: Cranial cruciate ligament rupture (CCLR) results from a multifactorial degenerative process that leads to rupture of the ligament. Vector-borne pathogens (VBP) in dogs can induce joint disease but their role in CCLR has not been previously investigated. The aim of the present work is to evaluate the prevalence of VBP in dogs with CCLR. Methods: This was a prospective study that included 46 dogs presented for CCLR surgical treatment and 16 control dogs euthanized for diseases unrelated to the joints. Specimens collected included blood, synovial fluid, and syno‑ vial membrane biopsy. Pathogen testing consisted of serology for Leishmania infantum (quantitative ELISA), Ehrlichia canis/ewingii, Borrelia burgdorferi, Anaplasma phagocytophilum/platys, and Dirofilaria immitis (4DX IDEXX test), and PCR for L. infantum, Ehrlichia/Anaplasma spp., Bartonella spp., piroplasms (Babesia spp. and Theileria spp.), and filariae (D. immitis, Dirofilaria repens, Acanthocheilonema dracunculoides, Acanthocheilonema reconditum, and Cercopithifilaria spp.) on both EDTA-whole blood (EB) and synovial fluid (SF) samples. SF cytology and histopathological evaluation of synovial membrane were also performed. Results: The prevalence of VBP was 19.6% in the CCLR group and 18.8% in the control group, with no statistical difference among them. The presence of synovitis was not more frequent in CCLR dogs (45.6%) than in control dogs (43.7%). Lymphoplasmacytic infiltration was the most common inflammatory pattern detected in the joints of both groups of dogs. Conclusions: This study failed to demonstrate a role of canine VBP in CCLR or the presence or different pattern of joint inflammation in pathogen-positive dogs. Keywords: Leishmania, Ehrlichia, Anaplasma, Bartonella, Piroplasms, Filariae, Osteoarthritis Background involving genetics, anatomic conformation, and chronic Cranial cruciate ligament rupture (CCLR) is a common joint inflammation, eventually leading to rupture of the cause of pelvic limb lameness in dogs that results most ligament and osteoarthritis [3, 4]. It has been suggested often from a degenerative process that leads to rupture that immunopathological mechanisms may be involved of the ligament. Several risk factors have been described, in the development of degenerative CCL lesions [5] and, such as age, breed, sex, neutering status, and weight [1, furthermore, lymphoplasmacytic synovitis is a common 2]. However, CCLR is likely to have a multifactorial origin finding in dogs with CCLR [6]. Therefore, two hypotheses have been proposed regarding the role of chronic synovi- tis in the development of cruciate ligament fiber damage *Correspondence: Xavier.Roura@uab.cat 4 Hospital Clínic Veterinari, Universitat Autònoma de Barcelona, Carrer de and, consequently, CCLR. The first is that synovitis is an l´Hospital s/n, 08193 Bellaterra, Spain early and primary event inducing progressive ligament Full list of author information is available at the end of the article fiber disruption [7]. The second is that several intrinsic © The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. 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Parasites & Vectors (2022) 15:105 Page 2 of 6 factors may induce minor fiber ruptures with subsequent Sampling induction of chronic synovitis that itself contributes to Blood was collected by cephalic or jugular venipuncture the degenerative changes in the ligament, culminating in from all dogs. Synovial fluid (SF) samples were collected CCLR [7]. immediately prior to the surgery or euthanasia, following Previous publications reported an increased bacterial anesthetic induction and aseptic preparation of the limb. load in synovial membrane biopsies of inflamed stifles of Smears were made immediately after collection, obtained dogs with CCLR in comparison with healthy stifles, sug- following cytocentrifugation, and 0.5 ml of synovial fluid gesting that environmental bacteria can induce persistent was placed in a tube containing calcium ethylenediami- chronic synovitis [8, 9]. Other kinds of pathogens such netetraacetic acid (EDTA). as vector-borne pathogens (VBP) can induce acute and Incisional synovial membrane biopsy specimens were chronic joint disease in dogs, but their role in CCLR has obtained from the affected joint of dogs with CCLR or not been thoroughly investigated [10–14]. A recent study from a random stifle joint of control dogs following cau- performed in Brazil reported that 91.3% of 46 dogs with domedial parapatellar arthrotomy (1 cm sample from leishmaniasis presented joint abnormalities observed each dog). Synovial biopsy samples were fixed in neutral on physical examination, radiography, and/or computed buffered 10% formalin and routinely processed for histo- tomography, but CCLR was not included in the list of logic examination. joint anomalies described [14]. Other VBP such as Ehr- Serum samples were archived and used for the detec- lichia canis, Anaplasma phagocytophilum, Rickettsia tion and quantification of organism-specific serology. rickettsii, Borrelia burgdorferi, Babesia canis, Bartonella Both EDTA-blood and EDTA-SF samples from all dogs vinsonii subsp. berkhoffii, and filariae have been also were stored frozen for posterior polymerase chain reac- linked to joint disease, such as polyarthritis, in dogs [4, tion (PCR) analysis. All serology and PCR analysis were 10, 15–24]. performed at the same time at the end of the study. This study aims to detect several VBP in owned dogs with CCLR, to determine if there is an association between the Serology presence of VBP and CCLR, and to identify the presence of ­(LEISCAN® enzyme-linked immunosorbent assay A quantitative enzyme-linked immunosorbent assay a specific inflammatory pattern in the synovial membrane of dogs with CCLR and VBP. [ELISA]) was performed for Leishmania infantum anti- body detection (IDEXX Barcelona, Spain), which has a cut-off of 0.55, with values > 0.55 considered posi- Methods tive. A commercial qualitative assay kit (SNAP 4DX Patient selection Plus IDEXX, Hoofddorp, Netherlands) was employed This prospective cohort study was conducted from June for the detection of E. canis/ewingii, B. burgdorferi, and 2016 until October 2019 in a referral veterinary hospital A. phagocytophilum/platys antibodies, and D. immitis located in the Mediterranean basin. Forty-six dogs pre- antigen. sented for CCLR surgical treatment and 16 control dogs that were humanely euthanized for suffering from differ- DNA extraction and PCR amplification ent serious diseases that did not affect the joints, were prospectively included during that period. DNA was extracted from 400 µl of both blood and SF Complete information obtained for the 46 dogs samples as previously described [25]. Collected samples, included age, sex, breed, body weight, general and ortho- previously defined as specific pathogen-free by PCR, were pedic examination, location of CCLR (unilateral or used as an extraction negative control in each extraction bilateral), and pre-anesthetic urine and blood analysis. batch. Samples were tested in an operator blind man- Furthermore, clinical reports for all dogs included in this ner, and piroplasms (Babesia spp. and Theileria spp.), study were reviewed to look for diagnosis of vector-borne Bartonella spp., Ehrlichia/Anaplasma spp., and filariae disease, before surgery or euthanasia, or at any time after [D. immitis, Dirofilaria repens, Acanthocheilonema dra- surgery until the end of the study (follow-up ranged from cunculoides, Acanthocheilonema reconditum, and Cer- 1.5 to 4.5 years). Owner consent was received for all dogs copithifilaria spp.] were targeted using genus-specific a total volume of 20 μl containing ­SYBR® Select Master prior to their enrolment in the study. This study was PCR assays [26]. Briefly, real-time PCR was carried out in carried out in accordance with ethical guidelines of the International Council for Laboratory Animal Science. Mix (Thermo Fisher Scientific), specific primer, and 4 μl of 1/5 diluted DNA. The thermal cycling profile was 50 °C for 2 min and 95 °C for 15 s and 60 °C for 1 min, and a Tabar et al. Parasites & Vectors (2022) 15:105 Page 3 of 6 dissociation curve added at the end of the run. Water was intact and two spayed) that ranged from 4 to 17 years in used as a PCR negative control and commercial DNAs as age, and 10 different breeds (Additional file 1: Table S1). positive PCR control. Quantitative L. infantum PCR was Reasons for euthanasia included neoplasia (five), chronic performed as described by Francino et al. [25]. Leishma- renal failure (two), gastric dilatation-volvulus (one), pitu- nia infantum DNA load was classified as very low, low, itary hyperadrenocorticism (one), discal hernia (two), medium, high, or very high load as reported by Martínez urethral obstruction (one), cardiac failure (two), refrac- et al. [27]. The eukaryotic 18S ribosomal ribonucleic acid tory epilepsy (one), and acute liver failure (one). There (rRNA) (Thermo Fisher Scientific) was used as an endog- was no previous clinical history, clinical signs, or clinico- enous control to ensure proper DNA extraction. pathological abnormalities consistent with VBP infection in any of the dogs included in this study. Sequencing The prevalence of VBP in the CCLR group was 19.6% In all cases in which SF or blood samples were PCR-posi- (9/46). Leishmania infantum was detected in six dogs, tive, direct DNA sequencing was performed to character- with three seropositive, one SF-PCR-positive, and two out using the ­BigDye® Terminator v3.1 Cycle Sequencing ize pathogens at the species level. Sequencing was carried both seropositive and SF-PCR-positive. Ehrlichia spp. were found in three dogs, with two seropositive and one Kit (Thermo Fisher Scientific) following the manufac- seropositive and blood-PCR-positive (E. canis). One of turer instructions and with the same primer used in the those dogs was co-infected with L. infantum (SF-PCR- PCR and sequences compared with the GenBank data- positive) and Ehrlichia spp. (seropositive). Finally, Theile- base (https://​blast.​ncbi.​nlm.​nih.​gov/​Blast.​cgi). ria equi DNA was detected in the SF sample of one dog (Additional file 2: Table S2). Cytologic and histologic examination Among the control group, the prevalence of canine Synovial fluid smears, obtained following cytocentrifu- VBP was 18.8% (3/16). All three dogs were positive for gation, were stained with a rapid modified Romanowsky L. infantum, with one seropositive, one blood-PCR-pos- stain (QUICK PANOPTIC, Química Clínica Aplicada, itive, and one seropositive and both blood- and SF-PCR- Amposta, Spain), and light microscopy examination was positive (Additional file 2: Table S2). performed only to detect the presence of pathogens. No VBP were detected by light microscopy in any Hematoxylin and eosin-stained biopsies of synovial SF-smear of any dog included in this study, and the membranes were evaluated by light microscopy. Samples total prevalence of VBP was not statistically different were evaluated for the presence and pattern of inflam- between dogs with CCLR and control dogs [odds ratio matory cells and characterized as lymphoplasmacytic, (OR) = 0.949, 95% confidence interval (CI) 0.22–4.05, granulomatous, neutrophilic, or mixed. Dogs were con- P = 0.629]. sidered to have synovitis if more than one inflammatory Unilateral CCLR was detected in 29 dogs (five of them cell/high-power field (hpf ) was identified. with VBP), while CCLR was bilateral in 17 dogs (four of them with VBP), meaning that the presence of bilateral Statistical analysis CCLR was not statistically more frequent among dogs Statistical analysis was performed using commercially with VBP (OR = 0.677, 95% CI 0.15–2.97, P = 0.439). available statistical software (IBM SPSS Statistics v.19). Histopathology of biopsies of synovial membranes Values for the prevalence of VBP (positivity to any of yielded synovitis in 45.6% (21/46) of dogs with CCLR, them) and for the presence of inflammation in joint cap- with various inflammatory patterns including lymphop- sule biopsy were established. Contingency table analysis lasmacytic (17), neutrophilic (one), granulomatous (one), was performed. Chi-square test and Fisher’s exact test and mixed lymphoplasmacytic and granulomatous (two); were used to compare proportions of positivity, and sta- and in 43.7% (7/16) of control dogs, all of them with lym- tistical significance was set at P-value ≤ 0.05. phoplasmacytic infiltration. Presence of synovitis was not statistically more frequent in dogs with CCLR compared Results with control dogs (χ2 = 0.017, df = 1, P = 0.895) or in dogs This study included 62 dogs, 46 with CCLR and 16 con- with or without VBP (χ2 = 0.141, df = 1, P = 0.708). Fur- trols. Among CCLR dogs there were 25 females (17 intact thermore, a different or specific inflammatory pattern and eight spayed) and 21 males (15 intact and six spayed), was not detected among dogs with VBP, independently of that ranged from 6 months to 11 years in age. There were whether they had CCLR or were control dogs (Additional 16 different breeds, with the most common being mon- file 2: Table S2). grel dogs (n = 18). In the control group there were eight When the outcome of VBP-positive dogs with CCLR females (four intact and four spayed) and eight males (six was reviewed, two of three dogs with L. infantum Tabar et al. Parasites & Vectors (2022) 15:105 Page 4 of 6 SF-PCR-positive, but without previous clinical history controversial [10, 15–17]. In the present study, three or diagnosis of Leishmania infection, developed clini- dogs with CCLR were found to have Ehrlichia antibod- cal signs consistent with patent leishmaniasis between ies or DNA. However, in the two only seropositive dogs, 9 to 12 months after CCLR surgery (Additional file 2: infection could not be confirmed, and perhaps it could Table S2). simply reflect exposure or past infection. The third dog, seropositive and E. canis PCR-positive, never developed Discussion any other clinical sign or laboratory abnormality consist- This study failed to demonstrate a role of VBP in CCLR ent with patent or subclinical ehrlichiosis, either before or the presence of a specific pattern of joint inflammation or after CCLR surgery. This could imply that this dog in VBP-positive dogs, even though several canine vector- perhaps was in an acute stage of the disease and could borne diseases have been associated with joint damage have recovered alone, or that it was in a subclinical stage. [4, 10, 14, 16–22, 24], and some of them, especially leish- Either of these two scenarios probably rules out a rela- maniasis, are considered endemic in the area where the tionship between CCLR and Ehrlichia infection. present study was performed [28]. Larger case–control Theileria equi is one of the equine piroplasms which is studies would likely be needed to clarify the role of vari- enzootic in Spain, with almost half of the horses having ous vector-borne organisms as a cause or cofactor in the antibodies or circulating parasitemia [34]. This parasite development of CCLR. has occasionally been detected in dogs, although its epi- In canine leishmaniasis, the frequency of orthopedic demiological and clinical significance remains unknown problems had been reported to range from 44.8% to as [35]. All the above, together with the fact that the dog high as 91.3%, when both orthopedic examination and in this study with T. equi in SF did not demonstrate any imaging (radiology and/or computed tomography) were other clinicopathological abnormalities throughout combined to look for joint abnormalities [14, 29]. Some the study period, could suggest that this pathogen was anomalies found in the orthopedic examination include opportunistic without clinical significance on CCLR. joint stiffness, lameness, soft tissue swelling, joint pain or Although Bartonella, A. phagocytophilum, B. burgdor- crepitation, and functional disability. Notably, dogs with feri, filariae, or other piroplasms such as Babesia have CCLR can have one or more of these described orthope- been associated with acute or chronic canine polyar- dic abnormalities, but in previous studies there was no thritis [10, 13, 17, 19–22, 36], no dog in this study was specific information about the prevalence of CCLR in positive for any of them. Those findings could be in con- dogs suffering from leishmaniasis. Theoretically, lame- cordance with the local geographic prevalence of those ness in leishmaniasis could be produced by polyarthritis, VBP reported in previous studies in the area evaluated with additional bone or muscle involvement, usually sec- in this study [16, 27, 34, 37]. However, it is worth noting ondary to inflammation associated with the deposition that the limitations regarding the sensitivity of the tech- of immune complexes within the joint because of a type niques used and the limitations associated with the speci- III hypersensitivity reaction [4, 12, 30]. However, primary mens collected for testing could also have contributed to joint infection could also occur, and parasites have been failure to detect these organisms. identified within macrophages by cytologic evaluation Lymphoplasmacytic arthritis was the most frequent of the synovial fluid and by histologic assessment of the histopathological finding in this study, in dogs both synovial membranes [31, 32]. Thus, infected dogs could with and without CCLR. This agrees with previous present with monoarthritis, oligoarthritis, or polyarthri- publications where lymphoplasmacytic synovitis has tis [18], and some reports indicate that stifle joint could been commonly described in dogs with CCLR [6], but be affected in close to 80% of cases [14]. In this study, L. it has also been detected in post-mortem samples from infantum was the most frequent VBP detected in dogs dogs without CCLR [7]. On the other hand, reactive with CCLR, although its prevalence was not significantly immune-mediated arthritis due to deposition of devel- different from that in control dogs, suggesting no role oped immune complexes secondary to VBP infection is of Leishmania infection in the pathogenesis of CCLR. A predominately neutrophilic [4, 11, 12, 18, 29]. This fact potential explanation for its detection in both groups of reinforces the idea that VBP did not play any role in the dogs could be the high prevalence of subclinical infection pathogenesis of CCLR, together with the fact that neither present in an endemic area of leishmaniasis [28, 33]. inflammatory pattern nor frequency of synovitis was sta- An association of polyarthritis with ehrlichiosis has tistically different between dogs with or without CCLR or been reported previously; however, there was no firm between dogs with or without VBP in this study. How- evidence to support it, and other possible co-infections ever, although not statistically significant, three dogs with were not ruled out, meaning that the relationship was CCLR in this study showed granulomatous synovitis, a Tabar et al. Parasites & Vectors (2022) 15:105 Page 5 of 6 kind of inflammation also reported in several tissues in Abbreviations CCLR: Cranial cruciate ligament rupture; VBP: Vector-borne pathogens; D: dogs with leishmaniasis [33, 38], but only one of them Dirofilaria; A: Acanthocheilonema; EB: EDTA-blood; SF: Synovial fluid; E: Ehrlichia; was positive for leishmaniasis. Although granulomatous L: Leishmania. inflammation is usually associated with the presence of Leishmania within the tissue [12, 13], amastigotes Supplementary Information could not be detected in this seropositive and Leishma- The online version contains supplementary material available at https://​doi.​ nia PCR-positive dog with granulomatous synovitis. The org/​10.​1186/​s13071-​022-​05205-x. cause of granulomatous inflammation in the other two dogs remains undetermined. Therefore, the current study Additional file 1: Table S1. Distribution of breeds among the study could not definitely rule out a potential role of this VBP groups. in the pathogenesis of synovitis and perhaps of CCLR in Additional file 2: Table S2. Dogs with vector-borne pathogens detected some of these dogs. (NA no abnormalities; LP lymphoplasmacytic; GR granulomatous; NIAD no This study has some limitations. The low number of infectious agent detected). dogs included, due to the difficulty in recruiting cases due to the strict inclusion criteria and the fact that it was Acknowledgements a prospective study with a control group, means that the the 16th ­CVBD® World Forum Symposium. The paper has been sponsored by Elanco Animal Health in the framework of statistical results must be taken with caution. Moreover, only dogs that received CCLR surgical treatment were Authors’ contributions included, excluding dogs initially diagnosed with VBP MDT and XR designed the study. MDT and JT were the clinicians who took the samples for the study and who were involved in the management of cases. and CCLR that ultimately did not undergo surgery. An LA performed the genetic tests and identified the final pathogen involved in additional limitation was the wide variety of diseases that the case report. CN performed the histological examination. MDT processed led to the euthanasia of control dogs and the fact that the database data and wrote the manuscript. XR was involved in drafting the manuscript. All authors read and approved the final manuscript. articular surfaces were not evaluated, especially knowing that the control population had a higher mean age. How- Funding ever, previous medical histories were extensively evalu- Not applicable. ated to exclude previous infections with VBP or diseases Availability of data and materials that could affect the joints. Furthermore, control dogs The datasets used and/or analyzed during the current study are available from were also included during the same period and area as the corresponding author on reasonable request. CCLR dogs, thus limiting the bias in the probabilities of VBP detection. Finally, the last limitation was the iden- Declarations tification of the presence of VBP. In this study we used Ethics approval and consent to participate serology, microscopy in SF cytology, and synovial mem- Collected data and samples used in this study were obtained from the brane biopsy, as well as PCR in blood and SF, seeking to diagnostic protocol and/or treatment of dogs included, under the verbal or written consent of the owners and in accordance with ethical guidelines of maximize the chances of VBP detection. However, the the International Council for Laboratory Animal Science. positive serologies were not conclusive of causality of the abnormalities found in the joint. Moreover, it was not Consent for publication Not applicable. possible to perform serology for all the pathogens sought for, nor could immunohistochemistry and/or PCR on the Competing interests biopsy be performed to increase the probability of detec- The authors declare that they have no competing interests. XR is a member of the CVBD World Forum. The CVBD World Forum was founded during the 1st tion of selected VBP. International CVBD Symposium from April 18th to 20th, 2006, in Billesley, UK, because of the increasing global threats through vector-borne diseases. The CVBD World Forum is supported by Elanco Animal Health. Conclusions Author details This study failed to demonstrate a role of several VBP in 1 Hospital Veterinario San Vicente Vetsum, Calle del Veterinario Manuel Isidro the pathogenesis of CCLR in dogs, or the presence or a Rodríguez García Nº17, San Vicente del Raspeig, 03690 Alicante, Spain. 2 IDEXX Laboratories, Carrer del Plom 2, 08038 Barcelona, Spain. 3 Vetgenomics, Parc de different pattern of joint inflammation in pathogen-pos- Recerca Universitat Autònoma de Barcelona, Edifici Eureka, 08193 Bellaterra, itive dogs. However, to overcome the limitations of this Spain. 4 Hospital Clínic Veterinari, Universitat Autònoma de Barcelona, Carrer de study, additional studies may be warranted to clarify the l´Hospital s/n, 08193 Bellaterra, Spain. potential relationship between VBP and CCLR in dogs. Received: 17 January 2022 Accepted: 14 February 2022 Tabar et al. 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Molecular identification of Bartonella species in