In most cases of inflammatory central nervous system [CNS] diseases in dogs, infectious agents remain undetected. Immunopathological studies suggest that such antigens may trigger an autoimmune response [“hit-and-Run hypothesis”] in some patients. In order to define the role of vector-borne pathogens in the aetiology of steroid-responsive meningitis-arteritis [SRMA] or meningoencephalomyelitis of unknown aetiology [MUE], blood and cerebrospinal fluid [CSF] of dogs were analysed for such pathogens. 66 client-owned dogs were included in the prospective multicenter study over a two year period. They were classified into 3 groups: 1] trauma group: dogs with non-inflammatory CNS diseases [n=21], 2] dogs with MUE [n=22], 3] dogs with SRMA [n=23].

DNA of A. phagocytophilum was found in EDTA-blood of 4 dogs [SRMA group]. Serological and PCR analyses for E. canis were negative in blood and serum of all dogs. B. henselae DNA was detected in blood of 1 dog [SRMA group]. There were no significant differences between the 3 groups regarding the seroprevalence of Bartonella spp. [n=61] and B. burgdorferi sensu lato [n=61]. Neither antibodies against TBEV in serum nor DNA of vector-transmitted agents was found in CSF in any of the dogs. Pasteurellaceae spp. DNA was detected in 3 dogs of the trauma group, suggesting contamination.

There was no correlation between the presence of E. canis or B. henselae DNA or elevated antibody titers against E. canis, Bartonella spp., TBEV or B. burgdorferi sensu lato and inflammatory CNS diseases. A. phagocytophilum may play a role as a trigger of a secondary immunopathy.

•    Central nervous system [CNS]
•    Inflammation
•    Meningoencephalitis of unknown aetiology [MUE]
•    Steroid-responsive meningitis-arteritis [SRMA]
•    Vector-borne microorganisms

Lazzerini K, Tipold A, Kornberg M, Silaghi C, Mietze A, et al. (2015) Testing for Vector-Transmitted Microorganisms in Dogs with Meningitis and Meningoencephalitis of Unknown Aetiology. J Vet Med Res 2(1): 1014.

CNS: central nervous system; SRMA: steroid-responsive meningitis-arteritis; MUE: meningoencephalitis of unknown etiology; CSF: cerebrospinal fluid; PCR: polymerase chain reaction; DNA: desoxyribonucleic acid; TBEV: tick-borne encephalitis virus; IFAT: immunofluorescence antibody test; ELISA: enzyme linked immunosorbent assay; MRI: magnetic resonance imaging; CT: computed tomography; EDTA: ethylenediaminetetraacetic acid; rRNA: ribosomal ribonucleic acid; NME: necrotizing meningoencephalitis; GME: granulomatous meningoencephalitis

Inflammatory lesions of the central nervous system [CNS] in dogs remain challenging for clinicians. Diseases causing CNS inflammation without a definitive diagnosis are a heterogeneous group currently known as meningoencephalitis of unknown aetiology [MUE] [1]. The pathogenesis of these diseases is still unknown, but a multifactorial aetiology with infectious, genetic, and immunopathological components may be responsible [2- 4]. Steroid-responsive meningitis-arteritis [SRMA] is another inflammatory CNS disease of unknown pathogenesis. It is considered an immune-mediated disease, although research to determine its aetiology is ongoing [5].

Vector-borne diseases, i.e. diseases caused by pathogens that are transmitted to vertebrate hosts by ectoparasitic vectors, are amongst the suspects to either directly or indirectly induce inflammatory diseases of the CNS [2,4,6]. The vectorborne pathogens Anaplasma phagocytophilum, Ehrlichia canis, Bartonella spp., Borrelia burgdorferi sensu lato, and tick-borne encephalitis virus [TBEV] have all been suspected to cause neurological signs in dogs [7-11].

A. phagocytophilum is the pathogen responsible for granulocytic anaplasmosis. Single cases of dogs with neurological signs have been PCR-positive for A. phagocytophilum in blood [12]. Other studies, however, have failed to confirm a correlation between elevated antibody titres against A. phagocytophilum and neurological deficits [13] or to detect the DNA of A. phagocytophilum in the blood or CSF of dogs with neurological signs [7].

Neurological signs such as ataxia and paraparesis have been described in the chronic form of canine monocytic ehrlichiosis, which is caused by E. canis [8]. A correlation between infection with E. canis and neurological signs remains unclear. Infections with Bartonella spp. are often subclinical but can lead to the development of clinical signs of canine bartonellosis. The most important species in dogs are Bartonella henselae [cat scratch disease] and B. vinsonii ssp. berkhoffii [14]. A few case reports have described dogs infected with Bartonella spp. that developed neurological signs of meningoradiculoneuritis [15] or meningoencephalitis [16,17]. The DNA of B. vinsonii ssp. berkhoffii was detected in the brain of one dog with granulomatous meningoencephalitis [GME] in a study of 109 dogs with neurological diseases [9].

Infection with B. burgdorferi sensu lato can also cause neurological signs in humans. To our knowledge, a correlation between infection with B. burgdorferi sensu lato [or any other Borrelia spp.] and encephalomyelitis in dogs has not yet been documented.

Tick-borne encephalitis [TBE] in Central Europe is caused by a flavivirus. If clinical signs appear, the disease generally has a fatal outcome within 4-7 days [11,18].

We analysed the blood and CSF of dogs to investigate the role of vector-borne microorganisms in the pathogenesis of SRMA and MUE.

Animals

We recruited 66 client-owned dogs between 12/2009 and 11/2011 presented to the Small Animal Clinic, Freie Universität Berlin; the Department of Small Animal Medicine and Surgery, University of Veterinary Medicine, Hannover; and the Small Animal Clinic in Trier. The patients were classified into three groups: trauma, MUE, and SRMA. The trauma group included 21 dogs with non-inflammatory CNS diseases [e.g. intervertebral disc disease], the MUE group included 22 dogs with meningoencephalitis, and the SRMA group included 23 dogs with SRMA. The number of patients was determined by animals that could be gathered in a period of two years with the established disease profiles and available samples

The criteria for inclusion were a diagnostic work-up, including CSF analysis and diagnostic imaging [magnetic resonance imaging [MRI] or computed tomography [CT] with intravenous contrast medium], and sufficient CSF, EDTA-treated blood, and serum available for further analysis. A presumptive diagnosis of MUE was declared when a dog showed neurological deficits, pleocytosis [>3 cells/µL] and elevated protein concentrations in the CSF, multifocal lesions with contrast enhancement on MRI or CT scans or no signs of neoplasia on the CT scan [4]. SRMA was diagnosed if the signalment, clinical, and neurological signs were consistent with the disease and if the CSF analysis showed polymorphonuclear pleocytosis [5]. Dogs with a low total nucleated-cell count were excluded unless they had a history of corticosteroid treatment before CSF collection [19-21]. Treatment with corticosteroids before CSF collection was documented for all cases (see Table 1). Five dogs of the MUE group, three dogs of the SRMA group, and 12 dogs of the trauma group had received corticosteroids prior to diagnostic testing (Table 1). Antibiotic treatment before CSF collection was documented for all cases (Table 1).

The number of dogs that originated from or had travelled to various geographical areas was documented (Table 2). Owner permissions were obtained for the use of the samples, and the study adhered to university guidelines.

DNA extraction and PCR analysis

PCR was performed on blood and CSF samples to detect the DNA of A. phagocytophilum, E. canis, and Bartonella spp. A qualitative eubacterial PCR was performed on CSF samples. Only dogs that originated from or had travelled to a foreign country where E. canis was endemic [Southern and Eastern Europe] and dogs with an unknown travel history were tested for E. canis [n=28].

A. phagocytophilum and E. canis

DNA extraction was performed using the High Pure PCR Template Preparation Kit [Roche Applied Science, Mannheim]. Real-time PCR was performed with HotStarTaq according to the manufacturer’s instructions [Applied Biosystems, Carlsbad] as previously described [22-24]. The target genes were msp2 for A. phagocytophilum and p30-10 for E. canis.

Bartonella spp.

The innuprep DNA Mini Kit was used for DNA extraction from EDTA-blood or CSF [Analytic Jena, Germany]. PCR was performed using the Mx3005P QPCR System according to the manufacturer’s instructions [target gene, gltA] [Stratagene, Heidelberg] as previously described [25]. The species were identified by restriction analysis of the PCR products [25]

Qualitative eubacterial PCR

A qualitative eubacterial PCR was performed on the CSF samples of 62/66 dogs to exclude the presence of other bacteria in the CSF. PCR targeting the highly conserved 16S rRNA gene was performed as previously described [26]. Amplicons corresponding to the 331-797 base-pair region of the Escherichia coli 16S rRNA gene were sequenced [single-stranded] by LGC Genomics GmbH, Berlin and were compared to nucleotide sequences from relevant databases [NCBI/BLAST].

Serological testing

Serological testing was performed to detect antibodies against TBEV [ELISA], E. canis [IFAT], B. burgdorferi sensu lato [IFAT, or C6 -ELISA if the IFAT antibody titre ≥1:128], and Bartonella spp. [ELISA]. Serum antibodies against TBEV were tested using the IgG All Species ELISA kit [Progen Biotechnik GmbH, Heidelberg]. The IFATs for E. canis and B. burgdorferi sensu lato [MegaScreen FLUO EHRLICHIA canis and MegaScreen FLUO BORRELIA canis test kits, MegaCor Diagnostik GmbH, Hörbranz] were performed following the manufacturer’s instructions. Titres of 1:64 or higher for E. canis were considered positive. The Borrelia IFAT is a qualitative method for detecting antibodies against B. burgdorferi sensu stricto, B. afzelii, and B. garinii. Sera with antiBorrelia antibody titres ≥1:128 in the IFAT were further analysed with a quantitative ELISA for the C6 peptide [IDEXX Laboratories, Ludwigsburg]. Titres below 1:128 were interpreted as negative. The ELISA for antibodies against Bartonella spp. was performed as previously described [27].

Statistical analysis

Groups were compared with non-parametric tests [KruskalWallis and Fisher’s exact tests] [SPSS 17.0 for Windows, SPSS Inc., USA]. A P-value <0.05 was considered significant.

Table 1: Medication received prior to CSF collection for the dogs in the three groups MUE, SRMA, and trauma.

Abbreviations: MUE: meningoencephalitis of unknown aetiology; SRMA: steroid-responsive meningitis-arteritis

Table 2: Travel histories of the dogs in the three groups MUE, SRMA and trauma [The numbers of dogs that originated from or had travelled to the various geographical areas are documented].

PCR test results [CSF and blood]

PCR testing of the CSFs did not detect any amplification products specific for the vector-borne pathogens A. phagocytophilum, E. canis, or Bartonella spp. DNA of A. phagocytophilum was detected in the EDTA-blood of four dogs [all in the SRMA group, 17.4%]. The difference between the three groups was statistically significant [p=0,012]. No differences were detected amongst dogs from different regions of Germany [Berlin, Trier, or Hannover] or between dogs that had stayed in Germany and dogs that had travelled to Southern or Eastern Europe. The PCR analyses of blood for E. canis was negative for all dogs [n=28]. B. henselae DNA was detected in the blood of one dog [SRMA group], and Pasteurellaceae spp. DNA was detected in three dogs of the trauma group.

Serological results

None of the dogs had detectable antibody titres against TBEV or E. canis in the sera.

Fourteen of 61 dogs had B. burgdorferi sensu lato serum antibody titres ≥1:128 using IFAT [seroprevalence 22.9%]. The seroprevalences of B. burgdorferi sensu lato did not differ significantly amongst the three groups [MUE, SRMA, and trauma]. Sera of the 14 IFAT-positive dogs were tested for the C6 peptide using ELISA; the antibody titre was >10 U/mL in two dogs of the MUE group and in one dog of the SRMA group. The titre levels established with the IFAT did not correlate with the C6 titres.

The antibody titre against Bartonella spp. in sera was measured in 61 dogs using ELISA. The seroprevalence averaged 83.6%. The three groups did not differ significantly amongst dogs from different regions of Germany [Berlin, Trier, and Hannover] or between dogs that had stayed in Germany and those that had travelled to Southern or Eastern Europe.

The blood and CSFs of dogs with SRMA and MUE were analysed for vector-transmitted microorganisms. The results were negative in the majority of cases. The negative PCR analyses in CSF corroborated the results of a previous study [Barber et al., 2010] that tested the CSFs from 74 dogs with neurological signs. DNA of E. canis, A. phagocytophilum, spotted fever group Rickettsia, Bartonella spp., and Borrelia spp. was not detected in these samples.

A. phagocytophilum DNA was not detected by PCR in the blood and CSF of dogs with neurological deficits in another study [7]. The DNA of A. phagocytophilum in the CSF, however, may have remained undetected due to a bacterial load in the brain below the limit of detection or to suboptimal PCR conditions.

In our study, DNA of Bartonella spp. was not detected in CSF, which can be due to a low bacterial load in dogs [28,29]. Brain tissues from 35 dogs were evaluated by PCR, and the DNA of B.

henselae was detected in the brain of only one dog with GME [9]. Future studies should thus employ pre-enrichment culture methods [28,30] for the detection of Bartonella spp. in CSF.

PCR testing of blood and CSF for E. canis DNA and antibody testing in sera were negative in all dogs tested [29/66 dogs had travelled to Southern, Northern, or Eastern Europe or had an unknown travel history and were therefore tested], corroborating the results of a previous study [9].

Qualitative eubacterial PCR was performed on the CSFs of 62/66 dogs. The DNA of Pasteurellaceae sp. was detected in the CSF of three dogs [trauma group]. Species identification was not successful in any of the cases. Most species of Pasteurellaceae are well adapted to mucosae, so contamination with oral Pasteurellaceae cannot be ruled out. These bacteria are not likely to play a relevant role in CNS inflammation.

PCR analyses for A. phagocytophilum DNA in blood were positive in 4/65 patients. All dogs belonged to the SRMA group [prevalence 17.4%]. Further information about these patients is listed in Table 3. Canine anaplasmosis and SRMA have overlapping clinical features [e.g. fever]. None of the dogs showed typical laboratory abnormalities of anaplasmosis [e.g. thrombocytopenia]. These results may imply that infection with A. phagocytophilum can play a role in the pathogenesis of immune-mediated CNS inflammation. The hit-and-run hypothesis describes such a phenomenon [31]. When an infectious agent induces a CNS inflammatory response, e.g. by molecular mimicry, the response can lead to clinical signs after the infectious agent has been eliminated or has not even entered the CNS [32]. A. phagocytophilum infections can also lead to secondary immunemediated processes such as immune-mediated thrombocytopenia or anaemia and polyarthritis. Platelet-bound antibodies were detected in humans [Wong and Thomas, 1998] and in dogs with granulocytic anaplasmosis [60-80%] [33,34], and two dogs with granulocytic anaplasmosis had a positive Coombs’ test [35].

The DNA of B. henselae was detected in the EDTA-blood of one patient [SRMA group]. In a previous study in Germany using the same method, the prevalence of B. henselae in dogs determined by PCR [of blood] was much higher than in published prevalence studies in developed countries [30]. The positive PCR in our study thus does not necessarily imply a causal relationship between infection and the development of neurological signs.

Antibody titres against Bartonella spp. were measured in sera using ELISA. Seroprevalences varied between 73.3% [MUE group] and 90.5% [trauma group]. The seroprevalence for the entire cohort was 83.6%, which was much higher than previously reported seroprevalences [36]. In our study, seroreactivity to various Bartonella spp. was tested. We do not know if the dogs were asymptomatic carriers of non-pathogenic strains or if the three groups were infected with pathogenic species, but seroprevalence did not differ significantly amongst the three groups. Fourteen of 61 dogs had IFAT antibody titres against B. burgdorferi sensu lato ≥1:128 [seroprevalence 22.9%], with no significant differences amongst the groups. Seropositivity is insufficient to determine causality between infection and development of clinical signs. Serologic testing for the C6 peptide can differentiate between naturally infected and vaccinated dogs. A C6 -ELISA was performed in 13 of the 14 dogs with elevated IFAT titres. Two dogs of the MUE group and one dog of the SRMA group had elevated C6 titres. We cannot exclude an association between the development of CNS inflammation and seropositivity for B. burgdorferi sensu lato, although a previous experimental study did not demonstrate that infection with B. burgdorferi sensu lato led to inflammatory disease of the CNS [10]. The evaluation of CSF for antibodies might provide more information about clinical relevance [10]. In a study in Sweden, all CSF samples from dogs with neurological symptoms were negative for A. phagocytophilum and B. burgdorferi sensu lato antibodies and DNA, although many of the dogs had serum antibodies for both agents [7]. In the present study, insufficient CSF was available for further analysis.

Serologic testing for antibodies against TBEV in sera was negative in all patients, which was not unexpected. More than 90% of all notified TBE cases have been in Bavaria, BadenWürttemberg, and Southern Hesse. The three clinics involved in the present study do not receive many patients from these areas. The travel histories of the dogs in our study (see Table 2) and the current known distribution of TBEV in Europe suggest the possibility of finding dogs with a previous infection with TBEV [11,37,38].

The diagnosis of the relevant pathogens could be enhanced by the measurement of paired antibody titres in sera [e.g. for Bartonella spp. and A. phagocytophilum]. A 4-fold increase in antibody titres is highly suggestive of an active infection. In the present study, however, second serum samples were not available for analysis.

A limitation of this study was that five dogs [one in the MUE group and four in the SRMA group] were pre-treated with doxycycline prior to referral and CSF collection. The pretreatment with doxycycline in these five cases may have led to false negative PCRs for the pathogens tested [39]. Interestingly, one of these patients was PCR-positive for A. phagocytophilum, which could have been due to a low dosage of the medication or to an inappropriate administration by the owner. All antibiotics can potentially cross a blood-brain barrier disrupted by inflammation. Antibiotic treatment with penicillin derivatives [12], fluoroquinolones [11], cephalosporin [1], or clindamycin [1] prior to CSF examination may have led to false negatives in the eubacterial PCR analysis.

In this study, the MUE group included a heterogeneous collection of inflammatory CNS diseases. In similar studies with an emphasis on pathological features, post-mortem examinations were available, and inflammatory disease states were further differentiated. In one study, 75 cases of GME, NME, or MUE were evaluated by PCR for vector-borne agents [from brain tissue or CSF], and the DNA of B. henselae was detected in only one dog [9]. A limitation of pathological studies is that examinations are conducted in the late stages of disease when significant tissue inflammation has already occurred and possible infectious triggers may have been eliminated. The current clinically oriented study focused on CNS inflammation rather than exact histological classifications of disease. This focus allowed us to achieve adequate group sizes and to examine living animals before the final stage of the disease. Examining the presence of pathogens in an earlier stage of inflammation may have increased the likelihood of their detection, assuming they play a role as triggers of immune responses as stipulated in the hitand-run hypothesis [40]. These negative results from living and diseased [or dead] animals indicate that extended experimental studies with an emphasis on immunopathological processes are needed to determine the potential immunogenic influence of vector-borne pathogens on CNS inflammation.

Table 3: Description of patients PCR-positive for A. phagocytophilum in blood.

Abbreviations: Hct: haematocrit; PLT: platelet count; TNCC: total nucleated-cell count; WBC: white blood cells; MRI: magnetic resonance imaging; SRMA: steroid-responsive meningitis-arteritis

E. canis and TBEV appear to be rare causes of inflammatory CNS disease. Neither the DNA of E. canis nor antibodies against either pathogen were detected in dogs with MUE or SRMA from various regions of Germany. B. burgdorferi sensu lato is an unlikely cause of inflammatory CNS diseases in dogs. Infection with Bartonella spp. was not correlated with and MUE or SRMA. A. phagocytophilum, however, may play a role as a trigger of a secondary immunopathy.

We would like to thank all members of the clinical staff for helping with patient care and treatment, especially Miss Ariana Maiolini for her support.

Conflict of interest

The authors certify that they have no affiliations with or involvement in any organisation or entity with any financial interest [such as honoraria; educational grants; participation in speakers’ bureaus; membership, employment, consultancies, stock ownership, or other equity interest; and expert testimony or patent-licensing arrangements] or non-financial interest [such as personal or professional relationships, affiliations, knowledge, or beliefs] in the subject matter or materials discussed in this manuscript.

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