Clinical and Diagnostic Features in Three Dogs Naturally Infected with Borrelia spp

The aim of this study was to present clinical and neurological signs, laboratory abnormalities, serologic and/or molecular findings in three dogs from the region of Brno in the Czech Republic. All dogs were naturally infected with Borrelia burgdorferi sensu lato. The evidence of borrelial infection was proved by serial blood sampling for IgM and IgG anti-borrelial antibodies or plasma PCR. The dogs manifested corresponding clinical signs and one or more of the following criteria were fulfilled: (1) 4-fold or greater increase or decrease in B. burgdorferi s. l. IgM or IgG antibodies serial titres in acute and convalescent stage of infection, (2) a shift from positive IgM to IgG antibodies titres, (3) decrease of IgM with concurrent increase of IgG antibodies in serial titres, (4) detection of borrelial DNA by PCR. Other possible tick-borne infections were excluded. All three dogs showed neurological signs (two of them meningoencephalomyelitis, one seizure connected with progressive renal disease). Their history, clinical signs, diagnostic procedures and treatment are described. Two of the dogs died and only one with meningoencephalomyelitis survived. Our results show that borrelial infection must be taken into consideration, not only in cases with febrile and orthopaedic signs but also in many other clinical syndromes. Borrelial infection, meningoencephalomyelitis, PCR, seizure, renal disease Borreliosis is a zoonotic tick-borne disease caused by a Gram-negative spirochete Borrelia burgdorferi sensu lato (B. burgdorferi s.l.) which includes a complex of genospecies, three of which are considered to be pathogenic in dogs: B. burgdorferi sensu stricto (B. burgdorferi s. s.), Borrelia garinii (B. garinii) and Borrelia afzelii (B. afzelii) (Hovius et al. 1999a Hovius 2005 Greene and Straubinger 2006). The main European vector is the tick Ixodes ricinus. Many species of mammals and birds were recognized as a reservoir of B. burgdorferi s. l. (Gern et al. 1998 Hulinska et al. 2002 Piesman and Gern 2004). The clinical form of borreliosis occurs in humans, domestic animals, especially dogs, horses and cattle (Burges et al. 1987 Greene et al. 1991 Cohen et al. 1992 Skotarczak et al. 2005 Kybicová et al. 2009). The close contact between dogs and humans, the common environment and the fact that borreliosis is emerging even in cities can be considered as indicators for outbreak detection (Štefančíková et al. 1998 Goossens et al. 2001). Clinical signs depend on the individual host response and vary widely, developing in relatively few individuals (Levy and Dreesen 1992 Levy and Magnarelli 1992). Both in humans and dogs, the condition can cause dermatological, musculoskeletal, neurological, renal and cardiovascular signs (Greene et al. 1991 Azuma et al. 1993 Straubinger et al. 1997 Straubinger 2000 Straubinger et al. 2000 Skotarczak and Wodecka 2003 Skotarczak et al. 2005 Greene and Straubinger 2006). ACTA VET. BRNO 2010, 79: 319-327; doi:10.2754/avb201079020319 Address for correspondence: Pavel Schánilec Small Animal Clinic University of Veterinary and Pharmaceutical Sciences brno Palackého 1/3, 612 42 Brno, Czech Republic Phone: +420 541 562 387 E-mail: [email protected] http://www.vfu.cz/acta-vet/actavet.htm The diagnosis is based on a combination of several factors: epidemiology-epizootiological information, clinical signs, serological tests and PCR (Straubinger 2000 Straubinger et al. 2000 Skotarczak and Wodecka 2003 Skotarczak et al. 2005 Pejchalova et al. 2006 Kybicova et al. 2009). The seroprevalence for B. burgdorferi s. l. in dogs was assessed in many European countries, e.g. in Slovakia (Štefančíková et al. 1998), Poland (Skotarczak et al. 2005), and Sweden (Egenvall et al. 2000). Specific antibodies to B. burgdorferi s. l. were detected in 6.5% of the population in the Czech Republic and the seroprevalence ranged between 0.0% and 28.6% (Pejchalová et al. 2006). In a recent study also in the Czech Republic the seropositivity of ELISA in IgM and IgG was 2.4% and 10.3%, respectively (Kybicová et al. 2009). In the Czech Republic DNA of B. garinii was detected in a blood sample of a dog only in one case (Kybicová et al. 2009). The aim of this work is to present three cases of dog patients with a variety of clinical signs and positive detection of borrelial infection by serological tests or PCR. Materials and Methods All patients were presented to the emergency service of the Small Animal Clinic of the University of Veterinary and Pharmaceutical Sciences Brno and hospitalized there. All dogs were regularly vaccinated with polyvalent vaccines and dewormed but none of them was vaccinated against borreliosis. All dogs suffered from severe tick infestation and all dogs came from Brno/South Moravia, an endemic area of tick-borne encephalitis and borreliosis (Klimeš et al. 2001 Pejchalova et al. 2006). Laboratory criteria for assessment of borrelial diagnosis Patients were diagnosed with borrelial infection if they manifested corresponding clinical signs and one or more of the following criteria were fulfilled: (1) 4-fold or greater increase or decrease in B. burgdorferi s. l. IgM or IgG antibodies serial titres in acute and convalescent stage of infection, (2) a shift from positive IgM to IgG antibodies titres, (3) decrease of IgM with concurrent increase of IgG antibodies in serial titres, (4) detection of borrelial DNA by PCR. CSF examination, haematology and serum biochemistry Complete CSF analysis was made within 30 min from collection (Bagley 2003 Bagley and Bohn 2003 Bohn and Bagley 2003). Cell counts were performed using the Fuchs-Rosenthal counting chamber. Blood and cerebrospinal fluid (CSF) smears were stained with Hemacolor (Merck KGaA, Darmstadt, Germany). Routine haematological examination and selected biochemical values was performed. Serology and PCR The sera were examined by the enzyme immunoassay (EIA) for detection of borrelial IgG and IgM antibodies (TEST-LINE, Brno, Czech Republic), by EIA for the detection of tick-borne encephalitis virus (TBEV) Ig antibodies (TEST-LINE, Brno, Czech Republic) and by A. phagocytophilum immunofluorescence (IFA) canine IgG antibody test (Fuller Laboratories, Fullerton, CA, USA), all according to manufacturer’s instructions. Levels of index positivity of antiborrelial antibodies: < 0.85 negative, 0.85-1.15 dubious, > 1.15 positive. The DNA samples were isolated from blood and CSF and were analyzed by standard PCR (Kybicová et al. 2009). Positive DNA samples were retested by restriction fragment length polymorphism (RFLP) analysis with 5S (rrfA)-23S (rrlB) rDNA intergenic spacer primers (Derdáková et al. 2003) as in Kybicová et al. (2008). Case studies Case 1 (5–7/ 2002) A five-month-old dog, 13 kg, intact male Nova Scotia Duck Tolling Retriever was presented with a 2-week history of inability to move, general hyperesthesia and spasticity of the muscles of the head and neck. The previous veterinarian collected blood samples (day -14) for borreliosis (see Table 2) because he found the dog pyretic (3 previous days) and treated him with high doses of co-amoxicillin (30 mg·kg-1 PO BID) and non-steroidal drugs. The condition of the dog improved over the next 3 days. A week later, the dog started refusing to lie down showing kyphosis, often remaining in a sitting position, being unable to make head and neck ventroflexion. At admission (day 0), the dog showed generalized hyperesthesia including vocalization and inability to move. The mental status alternated from depression and stupor to hysteria, accompanied by episodes of opisthotonus and myoclonia of both forelimbs. The cervical muscles were hypertonic and strong pain was elicited with mild manipulation of the neck. Examination of cranial nerves revealed moderate decreased trigeminal (CN V) sensation and facial (CN VII) reactivity on the left side with absence of reactions for the same nerves on the right side. Spinal reflexes showed hyperreflexia in all of four limbs. The dog remained preferentially in right lateral recumbency. CBC (Table 1) and antinuclear antibody test (ANA test) were sampled. Standard fluid support and treatment with co-amoxicillin (25 mg·kg-1 IV TID) was given, together with Diazepam (single dose 1.5 mg·kg-1 IV slowly) followed by phenobarbital (2 mg·kg-1 IV BID -TID) to control mental status. 320