Detection of Borrelia burgdorferi DNA in tissues from dogs with presumptive Lyme borreliosis

OBJECTIVE: To develop a quantitative PCR assay for detection of Borrelia burgdorferi DNA in formalin-fixed, paraffin-embedded tissues; compare results of this assay with results of immunohistochemical staining of tissues from seropositive dogs; and determine whether B burgdorferi DNA could be detected in renal tissues from dogs with presumptive Lyme nephritis. DESIGN: Cohort study. SAMPLE POPULATION: Archived tissue samples from 58 dogs. PROCEDURES: A quantitative PCR assay was performed on formalin-fixed, paraffin-embedded tissue sections from the dogs. Results were compared with results of immunohistochemical staining, B burgdorferi serostatus, clinical signs, and necropsy findings. RESULTS: 38 dogs were classified as having positive or equivocal results for Lyme borreliosis, and 20 were classified as having negative results on the basis of clinical signs, serologic findings, and pathologic abnormalities. Borrelia burgdorferi DNA was amplified from tissue samples from only 4 (7%) dogs, all of which had been classified as having positive or equivocal results for Lyme borreliosis and had signs of presumptive Lyme nephritis. Results of PCR assays of renal tissue were positive for only 1 dog, and there was no agreement between results of immunohistochemical staining (ie, detection of B burgdorferi antigen) and results of the PCR assay (ie, detection of B burgdorferi DNA) for renal tissues. CONCLUSIONS AND CLINICAL RELEVANCE: Results indicated that detection of B burgdorferi DNA in formalin-fixed, paraffin-embedded tissues is feasible, but that intact B burgdorferi DNA is rarely found in tissues from naturally infected dogs, even tissues from dogs with presumptive Lyme borreliosis. Further, findings support the contention that Lyme nephritis may be a sterile, immune complex disease.     

Identification of Borrelia burgdorferi genospecies inducing Lyme disease in dogs from Western Poland

Canine Lyme borreliosis may be caused by three Borrelia burgdorferi sensu lato genospecies. The prevalence of infection by Borrelia species was determined by nested polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) with the enzyme Fsp4H I in the blood of dogs naturally infested by ticks in an endemic region of Poland. Blood samples were collected from 98 dogs of various breeds, delivered to the Veterinary Clinic in Szczecin (northwestern Poland) for various reasons. Nested PCR revealed the presence of DNA characteristic of only 1 genospecies, i.e. B. burgdorferi sensu stricto (s.s.), in all PCR-positive samples. Digestion of PCR products from a fragment of the fla gene amplified with primers FLA1 and FLA2 gave only one band pattern consistent with the pattern obtained from sequence analysis of the fla gene from a reference isolate of B. burgdorferi s.s. GeHo (X15660) from GenBank.     

Serological studies on the infection of dogs in Ontario with Borrelia burgdorferi, the etiological agent of Lyme disease

A serological study was undertaken to determine whether dogs in Ontario are being exposed to Borrelia burgdorferi, the etiological agent of Lyme disease. This study consisted of a survey of randomly selected dogs and testing of diagnostic submissions from candidate Lyme disease cases. The survey of 1,095 dogs, bled between January 1988 and August 1989, revealed a total of 65 (5.9%) enzyme-linked immunosorbent assay (ELISA) reactors, of which 22 had immuno-fluorescent antibody assay (IFA) titers ≥1:32. All but one of the IFA-positive and 10 of the ELISA-positive, IFA-negative sera were further tested by western blot. Eight western blot positive and three equivocal reactors were obtained. Three of the eight confirmed reactors had visited areas known to be endemic for Lyme disease, leaving five reactors that might have been infected in previously undocumented areas for B. burgdorferi activity in Ontario. Diagnostic submissions of sera from 223 dogs were received between August 1987 and February 1992. Test results revealed 21 (9.4%) IFA reactors, of which only six had significant titers (≥1:256) and were reactive by an immunodot Borrelia test. All six dogs had travelled to known Lyme endemic areas. Based on results obtained from this study, it seems likely that the agent of Lyme disease is not widespread in Ontario.     

Search for Borrelia burgdorferi in kidneys of dogs with suspected "Lyme nephritis"

BACKGROUND: "Lyme nephritis" is a poorly characterized condition associated with proteinuria and often fatal renal failure in dogs with serological evidence of infection with Borrelia burgdorferi. OBJECTIVE: The aim of this study was to determine if intact B. burgdorferi organisms were present in the kidneys of serologically Lyme-positive dogs with histopathologic features of Lyme nephritis. ANIMALS: Twenty-six affected and 10 control dogs were identified over an 8-year period (1996-2004) in databases at Cornell University's College of Veterinary Medicine. Case inclusion required serologic evidence of natural exposure to B. burgdorferi and availability of renal tissue (frozen or paraffin embedded) exhibiting pathology consistent with Lyme nephritis. METHODS: Renal tissue samples were assessed using modified Steiner (silver) (MS) staining, immunohistochemistry (IHC), polymerase chain reaction (PCR) using 4 primer sets (eubacterial, B. burgdorferi, Bartonella, and canine genomic DNA), and fluorescence in situ hybridization (FISH) using a 5'-cy3-eubacterial probe for 16S rRNA. RESULTS: MS stain was positive in 1 case; IHC was negative in all cases. None of the B. burgdorferi or Bartonella PCR reactions was positive. Two of the B. burgdorferi FISH analyses were positive. CONCLUSIONS AND CLINICAL IMPORTANCE: Minimal evidence of the presence of intact B. burgdorferi or any other bacterial organism was found in the renal tissue of dogs with suspected Lyme nephritis. Direct renal invasion by B. burgdorferi organisms does not appear to be responsible for this syndrome.     

Borrelia burgdorferi Infection and Lyme Disease in North American Horses: A Consensus Statement

Borrelia burgdorferi infection is common in horses living in Lyme endemic areas and the geographic range for exposure is increasing. Morbidity after B. burgdorferi infection in horses is unknown. Documented, naturally occurring syndromes attributed to B. burgdorferi infection in horses include neuroborreliosis, uveitis, and cutaneous pseudolymphoma. Although other clinical signs such as lameness and stiffness are reported in horses, these are often not well documented. Diagnosis of Lyme disease is based on exposure to B. burgdorferi, cytology or histopathology of infected fluid or tissue and antigen detection. Treatment of Lyme disease in horses is similar to treatment of humans or small animals but treatment success might not be the same because of species differences in antimicrobial bioavailability and duration of infection before initiation of treatment. There are no approved equine label Lyme vaccines but there is strong evidence that proper vaccination could prevent infection in horses.     

Western immunoblot analysis for distinguishing vaccination and infection status with Borrelia burgdorferi (Lyme disease) in dogs.

Serodiagnosis of Lyme borreliosis in dogs is complicated by the use of commercially available Lyme disease vaccines that may cross-react with certain diagnostic assays. Western immunoblotting may be used to distinguish between dogs naturally exposed and those vaccinated against Borrelia burgdorferi. Because current vaccines are not 100% efficacious and dogs may be vaccinated after natural exposure, certain dogs may show serum antibody responses against both natural and vaccine exposure (dual status). In this study, samples from 17 nonexposed, 17 B. burgdorferi-bacterin vaccinated, 13 naturally exposed, and 8 dual-status dogs were tested by western immunoblot to determine if dual-status dogs could be reliably differentiated from naturally infected or vaccinated dogs. Reaction to outer surface protein A antigen of B. burgdorferi (31 kD) was a consistent marker for vaccination, appearing in all samples from vaccinate and dual-status dogs and in no samples from single-status naturally exposed dogs. Antibodies to 4 bands, at 80, 39, 29, and 28 kD, were present in all naturally infected and dual-status dogs. No samples from vaccinated or nonexposed dogs were reactive to all 4 of these bands simultaneously. Thus, vaccine and natural exposure produce differing antibody responses, whereas dual-status dogs produced the full antibody response of both types of exposure.     

Arthritis and systemic disease caused by Borrelia burgdorferi infection in a cow

Infection with Borrelia burgdorferi caused arthritis, myocarditis, glomerulonephritis, and pneumonitis in a cow. Spirochetes were detected by use of immunofluorescent staining in liver and lung specimens and were isolated from the liver. The carpal, stifle, and tarsal joints had marked synovial proliferation, and synovial fluid obtained from these joints had high antibody titers against B burgdorferi. The cow was from an area of Wisconsin that is not endemic for borreliosis.     

Arthritis caused by Borrelia burgdorferi in dogs

From October 1982 to May 1984, we studied 34 dogs from the Lyme, Conn area that had a history of tick exposure and lameness associated with pain, warmth, and/or swelling in one or more joints. Large numbers of polymorphonuclear leukocytes were seen in Giemsa-stained smears of synovial fluid from 9 dogs, and spirochetes (Borrelia burgdorferi) were found in 1 sample by darkfield microscopy and immunoperoxidase techniques. The geometric mean antibody titer to B burgdorferi in the 34 dogs was 1:2,700, compared with 1:285 in 43 clinically normal dogs from the same area (P less than 0.0001) and 1:50 in 29 dogs from an area in New Jersey that is not endemic for human Lyme disease (P less than 0.00001). We concluded that B burgdorferi in dogs may cause arthritis similar to that in human Lyme disease.     

Rheumatoid arthritis subsequent to Borrelia burgdorferi infection in two dogs

Two dogs with clinical signs of polyarthritis developed rheumatoid arthritis subsequent to Borrelia burgdorferi infection. In both dogs, the diagnosis of B burgdorferi infection was based on clinical signs of disease and high serum B burgdorferi titer. After antibiotic administration, both dogs had decreased B burgdorferi titer, but clinical response was temporary or was lacking. The dogs subsequently were rheumatoid factor-positive (antinuclear antibody- and anti-globulin-negative) and responded to anti-inflammatory drug administration. Development of rheumatoid arthritis in both dogs after B burgdorferi infection implicates the Borrelia organism as an infective agent leading to the development of rheumatoid arthritis in dogs. Dogs with clinical signs suggestive of B burgdorferi infection should have antiglobulin, anti-nuclear antibody rheumatoid factor, and B burgdorferi tests performed to aid definitive diagnosis.     

Complete Heart Block in a Dog Seropositive for Borrelia burgdorferi: Similarity to Human Lyme Carditis

Lyme disease has been recognized in humans since 1975 when it was associated with an outbreak of oligoarthritis in children in Lyme, Connecticut. Erythema chronicum migrans (ECM) is a clinical marker for the human disease, which usually appears within 3 to 32 days after an infected tick bite. Lyme disease is caused by spirochete, Borrelia burgdorferi, which is vectored by the hard ticks Ixodes dammini or Ixodes pacificus in the United States. In humans, Lyme disease has been found to cause a variety of clinical syndromes including cardiopathy, neuropathy, dermatopathy, and arthropathy. Human Lyme carditis is characterized by varying degrees of atrioventricular (AV) heart block that usually resolve regardless of therapy. Lyme disease has been reported in the dog as an arthropathy. This article reports a case of complete heart block and myocarditis in a dog with a positive titer for B burgdorferi, in which clinical and pathologic findings were similar to those seen in human Lyme myocarditis.     

采用PCR方法对我国蜱伯氏疏螺旋体感染的流行病学调查

通过调查我国已有的4种蜱伯氏疏螺旋体的感染情况,以进一步阐明莱姆病在中国的分布与流行状况。本研究通过筛选扩增伯氏疏螺旋体外膜蛋白A(OspA)基因片段的通用引物,获得1对特异性引物,优化反应条件后建立用于检测蜱体内伯氏疏螺旋体的PCR方法,其扩增片段大小为307 bp。该方法可检测出10 pg的阿氏疏螺旋体(Ba)、1 pg的伽氏疏螺旋体(Bg)和0.01 pg的狭义伯氏疏螺旋体(Bb)的3种不同基因型的伯氏疏螺旋体的OspA基因组DNA,表明其敏感性较好,适用于蜱感染伯氏疏螺旋体状况的调查。本研究从我国7个省采集到的667只蜱,进行伯氏疏螺旋体感染的流行病学调查,分类鉴定表明这些蜱分属革蜱属、血蜱属、牛蜱属和扇头蜱属。PCR检测所获数据表明它们的感染率分别为4%(10/264)、6%(11/137)、31.4%(59/185)和31%     

Evaluation of a canine C6 ELISA Lyme disease test for the determination of the infection status of cats naturally exposed to Borrelia burgdorferi

The efficacy of a commercially available in-office kit (SNAP 3Dx, IDEXX Laboratories) for detection of antibodies directed against an invariable region (IR6) of the B. burgdorferi surface protein VlsE (Vmp-like sequence, Expressed), a surface antigen of the spirochete recognized during active infection has been evaluated in dogs. The present study was conducted to determine whether this in-office test could be useful for detection of antibodies to B. burgdorferi in cats. Cats owned by clients of a veterinary hospital located in an area hyperendemic for Lyme disease were included in the study. When possible, cats with an outdoor lifestyle, bite wounds, or current tick infestation were recruited for the study to help ensure that animals with a likelihood of exposure to natural infection by B. burgdorferi would be included in the test group. Of the 24 cats tested, 17 samples were positive for antibodies to B. burgdorferi by the C6 ELISA kit. For all 17 of these samples, a duplicate sample tested by immunofluorescent assay (IFA) was in agreement with the ELISA. Five samples were negative by both assays. Two samples that were negative by the C6 ELISA test had low IFA titers (1:100). One of these two discrepant samples was negative and one was positive for antibodies to B. burgdorferi by the Western blot test. It was concluded that the C6 ELISA test performed with good agreement with the IFA and Western blot tests for detection of antibody to B. burgdorferi in the majority of cats tested. The test offers the advantages of producing a result rapidly (approximately 8 minutes), and it requires only two drops of serum, plasma, or whole blood.     

Shared flagellar epitopes of Borrelia burgdorferi and Borrelia anserina

Antigenic cross-reactivity between Borrelia burgdorferi and Borrelia anserina was studied using mouse immune sera and monoclonal antibodies. With immune sera, significant cross-reactivity between B. burgdorferi and B. anserina was demonstrated by indirect immunofluorescent assay. In immunoblots, most of the cross-reactivity was shown to be associated with the periplasmic flagella. Using monoclonal antibodies in immunoblots, it was shown that B. burgdorferi and B. anserina shared at least two flagellar epitopes, one of which was not shared with Borrelia hermsii or Borrelia coriaceae. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis profiles of whole cell lysates and the use of a species-specific monoclonal antibody (H5332) which reacts with a major outer surface protein (Osp A) of B. burgdorferi readily differentiated the two species at the molecular level.     

Borrelia burgdorferi infection in cats in the UK

Using an enzyme-linked immunosorbent assay (ELISA) and Western blotting techniques, cats from the north west of England and North Wales were tested for antibodies to Borrelia burgdorferi. Seropositivity to B burgdorferi in these cats was similar (4.8 per cent) to that found in dogs and horses in the UK from non-endemic areas. Cross-reactive antibodies to Leptospira interrogans serovars did not affect the cat B burgdorferi ELISA data. Clinical signs of Lyme disease were generally absent; lameness was rarely reported. As in other species, it must be considered that high levels of serum anti-borrelia antibodies are not diagnostic for clinical Lyme disease.     

Serum antibodies against particular antigens of Borrelia burgdorferi sensu stricto and their potential in the diagnosis of canine Lyme borreliosis

Dog sera (n = 118) were tested for antibodies recognizing Borrelia (B.) burgdorferi sensu stricto strain B31 (ATCC 35210) antigens. In total, 18 of the dog sera gave positive results in a whole cell sonicate ELISA (WCS ELISA). These positive sera were further evaluated by immunoblot assay, utilizing a whole bacterial lysate as antigens. 94.4% (17 of 18) of the dog sera reacted with immunodominant antigens at 20-22 kDa (protein C, pC), 31 kDa (outer surface protein A, OspA), 34 kDa (outer surface protein B, OspB), 41 kDa (flagellin), 60 kDa ("common antigen"), and/or 100 kDa (presumably p100). Sera recognizing pC (20-22 kDa) and antigens > 94 kDa always detected the highest number of antigen bands, indicating the specificity of those antigens in serological diagnosis. The results clearly demonstrate that the WCS ELISA is a useful tool for testing sera of dogs for antibodies against B. burgdorferi. However, positive results should be confirmed by immunoblot, using WCS as antigen. According to the presented data, we recommend criteria for B. burgdorferi immunoblots using dog sera as follows: sera have to be considered as positive if they detect the 41 kDa flagellin, and two of the 5 immunodominant antigens, namely > 94 kDa (presumably p100), 60 kDa ("common antigen"), 34 kDa and 29-31 kDa (OspB and OspA, respectively) and 20-22 kDa (pC). If sera only recognize the 41 kDa flagellin, this result is equivocal, requiring testing a second serum sample 4 to 8 weeks later.     

Evaluation of a commercial enzyme-linked immunosorbent assay for detection of Borrelia burgdorferi exposure in dogs

OBJECTIVE: To evaluate the effectiveness of a commercially available ELISA kit for detecting antibodies against Borrelia burgdorferi in dogs. SAMPLE POPULATION: Banked sera from 440 military working dogs were used for serologic analyses. PROCEDURE: Serum samples were analyzed for antibodies against B burgdorferi by use of a commercially available ELISA and subsequently by western blot analysis as a confirmatory test. RESULTS: Results from the ELISA indicated that 89 (20%) samples were positive for exposure to B burgdorferi or canine Lyme disease vaccine, and 351 (80%) were negative. Follow-up testing by western blot analysis indicated that results for 109 (25%) samples were positive and 331 (75%) were negative for exposure. All samples that had positive results on the ELISA also had positive results on western blot analysis (true positives). Of the 351 samples that had negative results on the ELISA, only 331 had negative results on western blot analysis (true negatives). The remaining 20 samples had positive results on western blot analysis. By use of a standard 2 x 2 table, it was determined that the ELISA had a sensitivity of 82%, specificity of 100%, positive predictive value of 100%, and negative predictive value of 94%. CONCLUSIONS AND CLINICAL RELEVANCE: The commercial ELISA kit evaluated in this study appeared to lack adequate sensitivity for detecting all potential cases of borreliosis in dogs. The ELISA was also unable to discriminate natural exposure from exposure attributable to vaccination, which could complicate interpretation of positive results and treatment of dogs with clinical signs.     

Class-specific and polyvalent enzyme-linked immunosorbent assays for detection of antibodies to Borrelia burgdorferi in equids

Class-specific and polyvalent ELISA were developed to detect IgM antibody or total immunoglobulins to Borrelia burgdorferi in equine sera. Analyses of 122 serum specimens, collected during 1985 from horses and ponies in tick-infested areas of Connecticut, revealed IgM antibody in 41 (34%) samples; titration end points ranged from 1:160 to 1:2,560. In polyvalent ELISA, 73 (16%) of 454 serum specimens contained IgM and/or IgG antibody. Seropositivity was highest (32%) for blood samples collected during May. Both ELISA procedures had comparable sensitivities.     

荧光定量PCR检测硬蜱体内莱姆病螺旋体的研究

为建立一个荧光定量PCR检测硬蜱体内莱姆病螺旋体的方法,根据GenBank登录的莱姆病螺旋体鞭毛蛋白FlaB序列,应用生物学软件进行序列比对,在保守的C段区设计与筛选特异引物和TaqMan探针。对荧光定量PCR反应体系与条件进行优化,验证方法的特异性、敏感性,并通过对感染螺旋体的蜱样本的检测,评价该方法的实用价值。结果显示,自然感染莱姆病螺旋体的35份蜱标本检测阳性符合率100%,正常蜱20份标本的检测结果均为阴性。该方法对牛巴贝斯原虫、泰勒原虫、边缘无浆体、金龟子绿僵菌、大肠杆菌等蜱体常见病原微生物所抽提的DNA的检测均呈阴性。荧光定量PCR方法检测质粒的灵敏度可达1×102拷贝/μL。TaqMan荧光定量PCR方法检测硬蜱体内莱姆病螺旋体具有较好的敏感性和特异性,可适于莱姆病的流行病学调查和监控。