Detection of vector-borne pathogens in dogs with cranial cruciate ligament rupture living in the Mediterranean region

Excerpts from a study by Maria‑Dolores Tabar, Javier Tabar, Carolina Naranjo, Laura Altet and Xavier Roura from 2022

https://doi.org/10.1186/s13071-022-05205-x

Translated and shortened by Sven Jan Arndt

Introduction

The detection of pathogens transmitted by vectors in dogs with cranial cruciate ligament rupture that live in the Mediterranean is of great importance for the health and well -being of animals. A precise examination and analysis of potential infections can be identified and suitable measures can be taken to contain the spread of diseases.

The role of vectors, such as ticks or fleas, in transmitting pathogens to dogs should not be underestimated. These tiny parasites can transmit various dangerous diseases such as Lyme disease, Ehrlichiosis or Leishmaniasis. Therefore, it is important to develop specific detection methods to identify these pathogens in dogs with cranial cruciate ligament rupture in the Mediterranean region.

An effective diagnosis requires a combination of different techniques and tests. These include, for example, serological tests, molecular examinations such as the polymerase chain reaction (PCR) or clinical examinations to identify symptoms and signs of infection. This extensive approach can be made precise diagnosis and adequate treatment can be initiated.

The detection of pathogens transmitted by vectors in dogs with cranial cruciate ligament rupture in the Mediterranean requires close cooperation between veterinarians, laboratories and research institutions. Only through joint efforts can we deepen the understanding of these diseases, develop preventive measures and improve the health of dogs.

Overall, the detection of pathogens transferred by vectors in dogs with cranial cruciate ligament rupture in the Mediterranean would be an important step to contain the spread of infections and ensure the well -being of the animals.

background

The rupture of the cranial cruciate ligament ( CCLR ) is a common cause of paralyzing the pelvic dimensions in dogs, which are usually due to a degenerative process that leads to the tore of the volume. Several risk factors have been described, such as age, breed, gender, castration status and weight [1,2]. However, it can be assumed that the CCLR has a multifactorial origin in which genetics, anatomical nature and chronic inflammation of the joints are involved, which ultimately leads to ligament tear and osteoarthritis [3, 4].

It was suspected that immunopathological mechanisms could be involved in the development of degenerative CCL lesions [5], and in addition, lymphoplasmic synovitis is a frequent finding in dogs with CCLR [6]. Therefore, two hypotheses on the role of chronic synovitis were set up in the development of cruciate ligament fiber damage and consequently the CCLR. The first hypothesis states that synovitis is an almost primary event that leads to a progressive destruction of the band fibers [7].

The second reason is that several inherent earlier publications reported on an increased bacterial burden on the synovial membrane in biopsies of inflamed knee joints of dogs with CCLR compared to healthy knee joints, which indicates that bacteria from the environment can trigger a persistent chronic synovitis [8, 9]. Other types of pathogens, such as For example, pathogens (VBP) transferred by vectors can trigger acute and chronic joint diseases in dogs, but their role in CCLR has not yet been thoroughly examined [10-14].

In a study recently carried out in Brazil, it was reported that 91.3 % of 46 dogs with leishmaniasis had joint anomalies that were found in physical examination, X -ray and/or in computer tomography, but the CCLR was not included in the list of the joint anomalies described [14]. Other VBP such as Ehrlichiacanis, anaplasma Phagocytophilum, Rickettsiarickettsii, Borrelia Burgdorferi, Babesia Canis, Bartonellavinsonii Sub. Berkhoffii and Filarios were also with joint diseases, such as B. polyarthritis, associated with dogs [4.10, 15-24].

The aim of this study is to demonstrate several VBP in dogs with CCLR, determine whether there is a connection between the presence of VBP and CCLR, and the presence of a specific inflammatory pattern in the synovial membrane of dogs with CCLR and VBP.

Methods

It was a prospective study in which 46 dogs took part in the surgical treatment of CCLR, as well as 16 control dogs that were put to sleep due to illnesses that had nothing to do with the joints. The removed samples included blood, synovial fluid and a synovial membrane biopsy.

The excited tests included the serology on Leishmania Infantum (Quantitative Elisa), Ehrlichiacanis/Ewingii, Borrelia Burgdorferi, Anaplasma Phagocytophilum/Platys and Dirofilaria Immitis (4DX IDEXX test) as well as PCR on L. Infantum, Ehrlichia/AnaPlasma SPP, Bartonella Spp., Piroplasms (Babesia Spp. and Theileria Spp.) And Filarien (D. Immitis, Dirofilaria Repens, Acanthocheilonema Dracunculoides, Acanthocheilonema Reconditum and Cercopithifilariasp.) In EDTA full blood (eb) and synovial fluid samples (SF). In addition, an SF cytology and a histopathological assessment of the synovial membrane were carried out.

Results

The study included 62 dogs, 46 with CCLR and 16 control dogs. 25 bitches (17 intact and eight neutered) and 21 males (15 intact and six neutered) at the age of 6 months to 11 years were affected by CCLR. It was 16 different breeds, the most common the mixed breed dogs (n = 18). In the control group there were eight bitches (four intacts and four neutered) and eight males (six intacts and two neutered) aged 4 to 17 years and 10 different breeds (additional file 1: Table S1).

The reasons for euthanasia included neoplasia (five), chronic kidney failure (two), magendilation volvulus (one), hypophyseshyperadrenokorticism (one), disco-hernia (two), urethral observation (one), heart failure (two), refractory epilepsy (one) and acute liver failure (one).

In none of the dogs included in this study there was a clinical history, clinical signs or clinical-pathological abnormalities that indicate a VBP infection. The prevalence of the VBP in the CCLR group was 19.6 % (9/46). Leishhmania Infantum has been demonstrated in six dogs, three of them seropositive, one SF-PCR positive and two both seropositive and SF-PCR positive. Ehrlichia spp. were detected in three dogs, two of them seropositive and one seropositive and blood-pccr positive (E. Canis).

One of these dogs was with L. Infantum (SF-PCR-positive) and Ehrlichia SPP. co -information (seropositive). Finally, the SF sample of a dog Theileriaequi-DNA was demonstrated (additional file 2: Table S2). In the control group, VBP's prevalence in dogs was 18.8 % (3/16). All three dogs were positive for L. Infantum, one of them seropositive, a blood PCR positive and a seropositive and both blood and SF-PCR positive (additional file 2: Table S2). No VBP was demonstrated by light microscopy in an SF smear of one of the dogs included in this study, and the overall prevalence of VBP did not differ statistically between dogs with CCLR and control dogs [ODDS ratio (OR) = 0.949, 95% confident interval (CI) 0.22-4,05, p = 0.629].

Unilateral CCLR was found in 29 dogs (five of them with VBP), while CCLR appeared bilaterally in 17 dogs (four of them with VBP), which means that the presence of a bilateral CCLR in dogs with VBP was not more common (OR = 0.677, 95% CI 0.15-2.97, p = 0.439). The histopathological examination of biopsies of the synovial membrane resulted in 45.6 % (21/46) with CCLR synovitis with various inflammatory patterns, including lymphoplasmacytic (17), neutrophil (one), granulomatous (one) and mixed lymphoplasmacytic and granulomatous (two); And with 43.7 % (7/16) of the control dogs, all of which had lymphoplasmacytic infiltration.

The presence of synovitis in dogs with CCLR was not statistically more frequent compared to control dogs (χ2 = 0.017, DF = 1, p = 0.895) or in dogs with or without VBP (χ2 = 0.141, DF = 1, p = 0.708). In addition, no different or specific inflammatory pattern was found in dogs with VBP, regardless of whether they had CCLR or control dogs (additional file 2: Table S2). When checking the results of VBP-positive dogs with CCLR, two of three dogs with L. Infantum-PCR-positive, but without prior clinical anamnesis or diagnosis of Leishmania infection, Between 9 and 12 months after the CCLR operation, clinical signs that indicate open leishmaniasis (Additional File 2: Table S2).

discussion

In this study, neither a role of the VBP could not be demonstrated in the CCLR nor the presence of a specific pattern of the joint inflammation in VBP-positive dogs, although several diseases transmitted by vectors were associated with dog damage [4, 10, 14, 16-22, 24] and some of them, in particular the leishmaniosis, in the area in which this study was carried out than apply endemic [28].

Larger case control studies would probably be necessary to clarify the role of various vector-handed organisms as the cause or cofactor in the development of CCLR. The dog's leismaniasis reported a frequency of orthopedic problems of 44.8 % up to 91.3 % if both orthopedic examination and imaging (radiology and/or computer tomography) were combined to search for joint anomalies [14, 29]. Anomalies that were found during the orthopedic examination include joint stiffness, lameness, soft tissue swelling, joint pain or crepitation and functional disabilities.

Dogs with CCLR can have one or more of these orthopedic anomalies described, but in previous studies there was no specific information about the prevalence of CCLR in dogs that suffer from leushmaniasis. Theoretically, the lameness in leishmaniasis could be caused by polyarthritis with additional bone or muscle involvement, usually secondary to inflammation, which is associated with the storage of immune complexes in the joint due to a hypersensitivity reaction of type III [4, 12, 30].

However, primary joint infection can also occur, and the parasites were identified in macrophages by cytological examination of the synovial fluid and through histological examination of the synovial membranes [31, 32]. Infected dogs can have monoarthritis, oligoarthritis or polyarthritis [18], and according to some reports, the knee joint could be affected in almost 80 % of the cases [14]. In this study, L. Infantum was the most common VBP, which was detected in dogs with CCLR, although their prevalence was not significantly different from that of the control dogs, which suggests that a leishmania infection in the pathogenesis of the CCLR plays no matter.

A possible explanation for the detection in both dog groups could be the high prevalence of a subclinical infection in an endemic leishmaniose area [28, 33].

A connection between polyarthritis and Ehrlichiosis has been reported earlier; However, there was no clear evidence of this, and other possible CO infections were not excluded, so the connection was controversial [10, 15-17]. In the present study, three dogs with CCLR Ehrlichia antibodies or DNA were demonstrated. In the two only seropositive dogs, however, the infection could not be confirmed, which may simply be due to exposure or an earlier infection. In the third dog, which was seropositive and E. Canis PCR-positive, there were no other clinical symptoms or laboratory anomalies before or after the CCLR operation that indicate open or subclinical honesty.

This could indicate that the dog was either at an acute stage of the disease and recovered on its own or that it was in a subclinical stage. Both scenarios are likely to rule a connection between CCLR and Ehrlum Infection. Heileria Equi is one of the horse piroplasms that is enzoo table in Spain, with almost half of the horses of antibodies or circulating parasitemia [34].

This parasite was occasionally also proven in dogs, but its epidemiological and clinical importance is still unknown [35]. All of this and the fact that the dog in this study with T. Equi in SF had no other clinical-pathological abnormalities during the entire study period could indicate that this pathogen was opportunistic and had no clinical importance for the CCLR.

Although Bartonella, A. Phagocytophilum, B. Burgdorferi, Filariae or other piroplasms such as Babesia were associated with acute or chronic hunde polyarthritis [10, 13, 17, 19-22, 36], there was no dog positive for one of these pathogens. These results could match the local geographical prevalence of this VBP, which was determined in previous studies in the area examined in this study [16, 27, 34, 37].

However, it should be noted that the limited sensitivity of the techniques used and the limitations associated with the samples taken for the study may also have contributed to the inability to detect these organisms. Lymphoplasmacytic arthritis was the most common histopathological finding in this study, both in dogs with and without CCLR. This is consistent with previous publications in which lymphoplasmacytic synovitis was commonly described in dogs with CCLR [6], but it has also been detected in postmortem samples from dogs without CCLR [7].

On the other hand, reactive immune-mediated arthritis is predominantly neutrophilic due to the deposition of developed immune complexes as a result of VBP infection [4, 11, 12, 18, 29]. This fact reinforces the idea that VBP plays no role in the pathogenesis of CCLR, along with the fact that neither the inflammatory pattern nor the frequency of synovitis was statistically different between dogs with or without CCLR or between dogs with or without VBP in this study .

Although not statistically significant, three dogs with CCLR in this study showed granulomatous synovitis, a type of inflammation also observed in several tissues of leishmaniasis patients [33, 38], but only one of these was positive for leishmaniasis. Although granulomatous inflammation is usually associated with the presence of Leishmania in tissue [12, 13], no amastigotes were detected in this seropositive and LeishmaniaPCR-positive dog with granulomatous synovitis.

The cause of the granulomatous inflammation in the other two dogs remains unclear. Therefore, in the current study, a possible role of these VBPs in the pathogenesis of synovitis and perhaps also CCLR in some of these dogs could not be definitively excluded. This study has some limitations. The small number of dogs included, due to the difficulties in recruiting cases due to the strict inclusion criteria and the fact that this was a prospective study with a control group, means that the statistical results should be treated with caution.

Additionally, only dogs that received surgical treatment for CCLR were included, so dogs that were initially diagnosed with VBP and CCLR and ultimately did not undergo surgery were excluded. Another limitation was the variety of diseases that led to the euthanasia of the control dogs and the fact that joint surfaces were not evaluated, particularly since the control population had a higher average age.

However, the medical history was comprehensively evaluated to exclude previous infections with ÖVPP or diseases that could affect the joints. Additionally, the control dogs were examined during the same time period and in the same area as the CCLR dogs, so the likelihood of detecting VBP was not as influenced. The final limitation concerned the determination of the presence of VBP. In this study, we used serology, microscopy in SF cytology and synovial membrane biopsy, as well as PCR in blood and SF to maximize the probability of VBP detection.

However, the positive serologies were not proof of the causality of the abnormalities found in the joint. Furthermore, it was not possible to perform serology for all pathogens sought, nor could immunohistochemistry and/or PCR be performed on the biopsy to increase the probability of detecting selected VBPs.

Conclusions

This study failed to demonstrate a role for different VBPs in the pathogenesis of CCLR in dogs nor the presence or different pattern of joint inflammation in pathogen-positive dogs. However, to overcome the limitations of this study, further studies may be required to clarify the possible association between VBP and CCLR in dogs.

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