A comparison of serologic tests for the detection of serum antibodies to whole-cell and recombinant Borrelia burgdorferi antigens in cattle

Serum samples from healthy dairy and beef cattle, living in tick-infested areas of Connecticut, USA, were analyzed by polyvalent enzyme-linked immunosorbent assays (ELISA), indirect fluorescent antibody (IFA) staining methods, or Western blot procedures to detect antibodies to tick-borne agents. Of the 80 sera tested by ELISA with whole-cell or 10 separate recombinant antigens (fusion proteins) of Borrelia burgdorferi sensu stricto, 57 (71%) were positive to 1 or more antigens, while 36 (45%) reacted to whole-cell antigens by IFA staining methods. Three (4%) of 80 samples had antibodies to Anaplasma phagocytophilum. There were antibodies to outer surface protein (Osp) A, OspB, OspC, OspE, OspF, protein (p) 41-G, p35, p37, and VlsE antigens of B. burgdorferi, but there was no reactivity to the p39 antigen by ELISA. Western immunoblots of a subset of 9 sera verified antibody presence in all samples and showed distinct reactivities to multiple proteins having molecular masses of about 31 kilodaltons (kDa), 34 kDa, 35 kDa, 41 kDa, and 83/93 kDa. High specificity (97%) was noted when 16 cattle sera containing antibodies to Leptospira interrogans serovars, Brucella sp., Anaplasma marginale, or A. phagocytophilum were tested by ELISA with separate whole-cell or recombinant B. burgdorferi antigens. An ELISA and Western blot analyses can be used to confirm the exposure of cattle to B. burgdorferi.     

Seroprevalence of antibodies against Borrelia burgdorferi and Anaplasma phagocytophilum in cats

OBJECTIVE: To determine whether cats in the northeastern United States develop serum antibodies against antigens of Borrelia burgdorferi and Anaplasma phagocytophilum and whether coinfection with the 2 organisms occurs. SAMPLE POPULATION: Serum samples from 84 healthy cats and 9 cats with lameness, fever, anorexia, or fatigue. PROCEDURE: Serum antibodies against B. burgdorferi and A. phagocytophilum were measured with an ELISA incorporating a whole-cell preparation or purified recombinant antigens, by means of Western blot analysis, or indirect fluorescent antibody (IFA) staining. RESULTS: ELISA results indicated that 44 of 93 (47%) sera contained antibodies against > or = 3 B. burgdorferi antigens, whereas 43 (46%) were reactive to whole-cell B. burgdorferi. Serum reactivity to protein 35, VlsE, and outer surface proteins A and F was most common. Seropositivity to > or = 3 antigens occurred at the same rate (5/9) in the 9 ill cats as in the 84 healthy cats (46% [39/84]). Of 13 sera reactive to recombinant antigens, 9 were seropositive as measured by Western blot testing with whole-cell antigen. Seropositivity rates of 30% and 38% were detected for antibodies against A phagocytophilum via IFA and ELISA testing, respectively. Fifteen (16%) sera had antibodies against both pathogens. CONCLUSIONS AND CLINICAL RELEVANCE: Cats living in areas infested by Ixodes scapularis ticks are exposed to B. burgdorferi and A. phagocytophilum and, in some instances, may be coinfected. Most cats appeared healthy. An ELISA incorporating specific recombinant antigens may be used adjunctively with Western blot and other assays to confirm B. burgdorferi and A. phagocytophilum infection in cats.     

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.     

Serologic confirmation of Ehrlichia equi and Borrelia burgdorferi infections in horses from the northeastern United States

OBJECTIVE: To determine whether horses living in tick-infested areas of northeastern United States with clinical signs of borreliosis or granulocytic ehrlichiosis had detectable serum antibodies to both Borrelia burgdorferi and Ehrlichia equi. DESIGN: Prospective study. ANIMALS: Serum samples from 51 clinically normal horses, 14 horses with clinical signs of borreliosis, and 17 horses with clinical signs of granulocytic ehrlichiosis. PROCEDURE: Serum B burgdorferi or E equi antibodies were measured by use of an ELISA, immunoblot analysis, or indirect fluorescent antibody (IFA) staining. RESULTS: Of the 82 serum samples tested, 37 (45.1%) and 13 (15.9%) had detectable antibodies to B burgdorferi or E equi, respectively. Test results indicated that 12 horses had been exposed to both agents, 11 of these horses had granulocytic ehrlichiosis. The ELISA regularly detected antibodies to the following recombinant protein (p) antigens of B burgdorferi: p29, p37, p39, and p41-G. The use of immunoblot analysis confirmed ELISA results by indicating antibody reactivities to antigens of whole-cell B burgdorferi having molecular masses of predominantly 31, 34, 37, 39, 41, 58, and 93 kd. CONCLUSIONS AND CLINICAL RELEVANCE: Horses living in areas where ticks (Ixodes scapularis) abound are sometimes exposed to multiple pathogens. Analyses for specific recombinant borrelial antibodies using an ELISA can help separate horses with borreliosis from those with granulocytic ehrlichiosis, even when antibodies to both etiologic agents are detected in serum samples. Analysis using immunoblots is sensitive, and along with ELISA or IFA procedures, is suitable for confirming a clinical diagnosis of each disease.     

Borrelia infections of ruminants in Europe

The occurrence of Borrelia spirochaetes in cattle in the Netherlands is reported for the second time, and in red deer and roe-deer in Austria for the first time. It is postulated that these spirochaetes are Borrelia burgdorferi rather than B. theileri. The reservoir role of ruminants in the epidemiology of human disease caused by B. burgdorferi in Europe should be investigated.     

Antibodies to Borrelia burgdorferi in New England horses: serologic survey

Twelve of 50 randomly selected horses from areas endemic for Borrelia burgdorferi had indirect fluorescent antibody titers of 1:8 to 1:2,048 against B burgdorferi. One of 50 horses from nonendemic areas had a titer of 1:8. This difference in the number of horses seropositive for B burgdorferi (P less than 0.002) and our finding that seropositive horses did not have agglutinating antibodies against potentially cross-reacting Leptospira spp indicated that horses in endemic areas were exposed to B burgdorferi and that the spirochete induced an antibody response in the horses.     

Clinical and serologic studies of canine borreliosis

During 1984 and 1985, blood samples were obtained from 271 dogs that were suspected of having borreliosis. The dogs lived in areas known to be infested with ticks and had been examined because of limb/joint disorders or for unknown illnesses marked by fever, anorexia, or fatigue. Lameness had been the most frequently reported clinical manifestation. Analyses of serum specimens, by an indirect fluorescent antibody (IFA) method or by an ELISA, detected antibodies to Borrelia burgdorferi, the etiologic agent of borreliosis in dogs and of Lyme disease in human beings. Antibody to B burgdorferi was detected in 76.3% of 114 specimens from dogs living in the lower Hudson Valley region of New York State (predominantly Westchester County), in 66.5% of 155 specimens from dogs from southern Connecticut, and in single specimens from dogs from Rhode Island and California. Geometric mean antibody titers peaked during the winter. Results of IFA tests and ELISA were in agreement, but the latter method yielded less variable results, had greater sensitivity, and was more easily standardized. Five dogs from New York State and Connecticut seropositive to B burgdorferi had developed kidney disorders during or after episodes of intermittent lameness. Application of murine monoclonal antibody in an IFA procedure verified the presence of B burgdorferi in renal cortical tissues from one dog.     

Efficacy of an amitraz-impregnated collar in preventing transmission of Borrelia burgdorferi by adult Ixodes scapularis to dogs

OBJECTIVE: To determine whether an amitraz-impregnated collar could prevent transmission of Borrelia burgdorferi by Ixodes scapularis to dogs. DESIGN: Laboratory trial. ANIMALS: 8 specific-pathogen-free Beagles. PROCEDURE: On days -15 and -1, all dogs had negative ELISA results for serum antibodies against B. burgdorferi. On day 0, 4 dogs were each fitted with an amitraz-impregnated (9%) collar, and 4 dogs served as untreated controls. On day 7, all dogs were infested with 100/scapularis (approx 50 females and 50 males) with a known B. burgdorferi infectivity rate of 39.4%. On days 21, 28, 35, 42, 56, 70, and 84, each dog was tested for serum antibodies against B. burgdorferi via ELISA and a western blot technique. Additional ELISA were also performed for serum antibodies against antigenically similar organisms. RESULTS: By day 70, all control dogs had developed serum ELISA responses ranging from 328 to 510 kinetics-ELISA units (equivalent to end-point titers of approx 43,500 to 60,000), whereas treated dogs remained seronegative throughout the study. Western blot assays performed on all serum samples confirmed that antibodies detected in control dogs reflected responses to specific antigens of B. burgdorferi, whereas treated dogs had no such antibodies. Additional serologic analyses confirmed that antibody responses observed in control dogs were not attributable to antigenically similar organisms. CONCLUSIONS AND CLINICAL RELEVANCE: Amitraz-impregnated collars prevented transmission of B. burgdorferi in 4 of 4 treated dogs and may be a useful management tool for prevention of borreliosis in dogs.     

Detection of antibodies to Borrelia burgdorferi in naturally infected horses in the USA by enzyme-linked immunosorbent assay using whole-cell and recombinant antigens

Blood samples were collected from 98 horses suspected of having borreliosis or granulocytic ehrlichiosis in Connecticut and New York State, USA during 1985, 1995, and 1996. Serum antibodies to Borrelia burgdorferi were detected by an enzyme-linked immunosorbent assay (ELISA), based on whole-cell and recombinant antigens, in 82 (84%) horses. Of the 181 sera tested, 59% were positive, using whole-cell antigens, compared to 48% with protein (p)37 and 35% with VlsE antigens. An ELISA containing either of these fusion proteins can be used as an adjunct to general screening by an ELISA or immunoblotting in animals not vaccinated for this disease.     

Encephalitis associated with Borrelia burgdorferi infection in a horse

Infection with Borrelia burgdorferi was associated with encephalitis in a horse. The horse lived in an area of Wisconsin endemic for B burgdorferi infection. Borrelia burgdorferi was isolated from the brain, but rabies virus was not detected in the brain. Serum obtained from the horse had a B burgdorferi antibody titer of 1:2,048, but was negative for antibodies to eastern and western encephalomyelitis.     

Experimental infection of laboratory mice and rabbits with several isolates of Borrelia burgdorferi sensu lato; comparison of antigens from different genospecies in serological measurement of immune responses

Infectivity of and immune responses to 28 Finnish Borrelia burgdorferi sensu lato isolates was studied in 3-4-week-old outbred NMRI and inbred BALB/c/Hy laboratory mice; rabbits were also inoculated. Twenty-one isolates were found to detectably infect mice. A variation among isolates in degree of infectivity was observed. Higher infection rate and higher average ELISA readings were recorded for intradermal than intraperitoneal inoculations. The results suggest differences between Borrelia genospecies in organotropism. The ear was frequently infected by representatives of all genospecies; among high infectivity experiments, this rate was highest, 100%, in infections by Borrelia afzelii. Further differences between genospecies specific organ distributions: B. burgdorferi sensu stricto and Borrelia garinii isolates seemed to infect the bladder relatively more frequently than B. afzelii did; B. afzelii isolates infected heart relatively more frequently than others did. Genospecies specific differences were demonstrated between antigens in reactivity, i.e. in their 'sensitivity' as reagents of ELISA and IFA methods to measure isolate specific immune responses. Antigens from two B. afzelii isolates differed clearly in sensitivity.     

Utility of an in-office C6 ELISA test kit for determination of infection status of dogs naturally exposed to Borrelia burgdorferi

Serologic evaluation for the diagnosis of Lyme disease has been confounded by several factors, including a high prevalence of clinically normal dogs testing seropositive, persistence of antibodies, and the introduction of vaccines that will induce antibodies detectable by immunofluorescent antibody assay, whole-cell ELISA, and Western blot assay. The utility of a commercially available in-office test kit (SNAP 3Dx, IDEXX Laboratories) for the simultaneous detection of Borrelia burgdorferi and Ehrlichia canis antibodies and Dirofilaria immitis antigen was evaluated for its ability to detect exposure to B. burgdorferi in both vaccinated and unvaccinated dogs from a highly Lyme-endemic area of Connecticut. The test kit is an ELISA that uses a synthetic peptide (C6) that duplicates the sequence of the IR6 region. The in-office C6 ELISA kit was found to be particularly useful in Lyme-endemic areas because it can be used conveniently and reliably in the clinic to determine a dog's infection status regardless of the vaccination history of the animal.     

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.     

Experimentally induced infection of cats with Borrelia burgdorferi.

To determine whether cats could be infected experimentally with Borrelia burgdorferi, 15 cats were inoculated with approximately 1,000 B burgdorferi. Seven cats were inoculated by the IV route, 2 by the oral route, 2 by the ocular route, and 4 by the oral-ocular route. Six control cats were inoculated with phosphate-buffered saline solution by the IV, oral, and ocular routes. Prior to the start of the study, all 21 cats were seronegative for B burgdorferi on the basis of results of the indirect fluorescent antibody (IFA) test, and their blood was B burgdorferi culture negative. All of the IV, orally, and ocularly inoculated cats developed IgG antibodies to B burgdorferi as detected by IFA testing. Of 4 oral-ocularly inoculated cats, 2 developed IFA-detectable antibodies and the remaining 2 cats developed low-titer response (1:128) on postinoculation (PI) day 10 only. All control cats remained seronegative. The organism was detected in blood smears from 2 of the IV inoculated cats on PI days 10 and 24 and from 2 oral-ocularly infected cats, 1 on PI days 17 and 24 and 1 on PI day 10. Spirochetes were not detected in the blood after PI day 24. The organism was isolated from tissues of only 1 cat (the lung of an ocularly inoculated cat necropsied at 7 months after inoculation). Spirochetes were not isolated from control cats. Neither clinical signs of infection nor gross or histologic abnormalities were found in any of the inoculated or control cats. Results indicate that cats are susceptible to infection with B burgdorferi, but clinically apparent disease may not be common.     

Use of a C6 ELISA test to evaluate the efficacy of a whole-cell bacterin for the prevention of naturally transmitted canine Borrelia burgdorferi infection

A commercially available C6 ELISA kit was used to detect antibodies induced by natural infection with Borrelia burgdorferi in dogs that lived in an area endemic for Lyme disease. Rates of infection were determined both for nonvaccinated dogs and those that had been vaccinated with a whole-cell B. burgdorferi bacterin (Lyme Vax, Fort Dodge Animal Health) before 6 months of age and were boostered annually. Vaccinated dogs had an infection rate of 5% (8 of 163), whereas 64% (25 of 39) of the non-vaccinated dogs were positive for B. burgdorferi antibodies. The preventable fraction, determined by comparing infection rates in unvaccinated and vaccinated dogs, was 92.2% (95% confidence interval: 84.3% to 96.3%). In addition, screening of nonvaccinated dogs at six Connecticut clinics (Middletown, Portland, Essex, Old Lyme, Durham, and Marlborough) with the C6 ELISA test revealed infection rates ranging from 41% to 73%, demonstrating a high level of infected dogs in the area. It was concluded that emphasis should be placed on vaccinating young dogs at risk for Lyme disease before they are exposed to infected ticks. Results of this study support the value of immunization with this whole-cell Lyme disease bacterin for dogs at risk for infection by B. burgdorferi.     

Serologic prevalence of Dirofilaria immitis,Ehrlichia canis,and Borrelia burgdorferi infections in Brazil

Dogs infected with Dirofilaria immitis, Ehrlichia canis, or Borrelia burgdorferi may show nonspecific clinical signs or may be asymptomatic. In Brazil, E. canis and D. immitis infections are frequently diagnosed based on the presence of classical signs; however, serologic tests are seldom performed to confirm the presence of infection. To estimate the seroprevalence of these three canine diseases in Brazil, 2,553 dogs presented at veterinary practices for various tests, routine treatments, or examinations were evaluated by an in-office commercial ELISA test kit (SNAP 3Dx, IDEXX Laboratories). Each dog was examined by the veterinarian, and a whole-blood sample was collected and immediately tested for the simultaneous detection of B. burgdorferi and E. canis antibodies and D. immitis antigen. D. immitis infection was detected in 51 dogs (2.0%) and E. canis antibodies were present in 505 dogs 19.8%). Only one dog tested positive for B. burgdorferi antibodies.     

Tick infestation and the prevalence of Borrelia burgdorferi and Babesia divergens in cattle in Bavaria

During the grazing period 2002 319 cattle from 31 farms located in 6 districts of southern Bavaria were examined for the presence of ticks in 4- to 5-week intervals, and 287 serum samples were tested for the presence of antibodies against Borrelia burgdorferi and Babesia divergens. Ticks were detected in all 31 farms with a mean prevalence of 69%. 3218 out of 3453 collected ticks were Ixodes ricinus; 139 nymphs, 19 larvae and 77 damaged adult specimens could only be determined to the Genus level (Ixodes).The seasonal pattern revealed the highest frequencies of ticks in May/June and September.The intensity of tick infestation of positive animals was generally low. 76.5% of parasitized cattle had 1-6 ticks per day of investigation. Individual cattle showed up to 250 ticks per day. The percentage of infested animals in each herd varied within the period between 0-100%. The examination of serum samples by immunofluorescence technique (IFAT) revealed positive anti-Borrelia antibody titers (> or = 1:64) for 45.6% of the animals. The within-farm seroprevalence of borreliosis ranged from 20 to 100% in 27 of the 31 farms. A significant correlation could be detected between the number of ticks/cattle and the anti-Borrelia burgdorferi IgG-titer. By contrast, there was no significant correlation between the age of the animals and anti-Borrelia serum titers. For comparative reasons, 64 IFAT-positive serum samples were tested by Western blot techniques for the presence of antibodies cross-reacting with Borrelia garinii antigen. These analyses revealed that 69% of the samples reacted positively, 28% were unclear and 3% were negative. Examinations of the 287 serum samples for the presence of anti-Babesia divergens antibodies revealed one positive animal with a titer of 1:16.     

A serological survey of Ehrlichia canis, Ehrlichia equi, Rickettsia rickettsii, and Borrelia burgdorferi in dogs in Oklahoma

Serum samples from 259 dogs were tested for antibodies to Ehrlichia canis, Ehrlichia equi, Rickettsia rickettsii, and Borrelia burgdorferi using the indirect fluorescent antibody test. The sera were obtained from submissions to the Oklahoma Animal Disease Diagnostic Laboratory during a 14-month period from June 1986 through July 1987. The rate for positive antibody titers to E. canis was 53%, to E. equi was 33%, to R. rickettsii was 38%, and to B. burgdorferi was 18%. Higher percentages of sera serologically positive to E. canis were found in the spring through the fall months, but there were no seasonal variations for E. equi, R. rickettsii, and B. burgdorferi. There was no consistent pattern of titers to the 4 antigens when age-groups of the dogs were compared. Forty-four different breeds were tested.     

Serological evidence of spirochaetal infections associated with digital dermatitis in dairy cattle

A potentially infectious aetiology for digital dermatitis in dairy cattle was investigated and centred on the possible involvement of spirochaetes. An enzyme-linked immunosorbent assay (ELISA) was developed to detect bovine anti-Borrelia burgdorferi (B31) and anti-Treponeme (USA bovine isolates) antibodies in the sera of cows; sera were further tested for antigen specificity by Western blotting. Compared to normal cows, those with digital dermatitis had a much higher seropositivity rate to B. burgdorferi and the treponemes. Significant correlations were shown between antibodies to B. burgdorferi and to Treponemes (P < 0.001), suggesting strong cross-reacting epitopes shared by these spirochaetes. In Western blotting of B. burgdorferi antigens, the main band detected by ELISA positive sera was the 41 kDa flagellar protein; lesser frequency of staining was seen with 34 (OspB), 39 and 55 kDa bands. For the USA treponeme antigens, ELISA positive sera gave reactions to the 34-kDa band and also bands at 41 and 55 kDa. Polyclonal antibodies to Treponema denticola and T. vincentii showed reactions with the bovine treponemes which were predominantly to the 34-kDa antigen. Monoclonal antibodies to B. burgdorferi flagella (41 kDa) antigen and OspA (31 kDa) did not detect any treponeme bands in Western blotting. The study has provided serological evidence that spirochaetes (which are related to human treponemes) may be involved in the pathogenesis of digital dermatitis.     

Competitive enzyme-linked immunosorbent assay for heartwater using monoclonal antibodies to a Cowdria ruminantium-specific 32-kilodalton protein

Hybridomas producing monoclonal antibodies (mAb) to Cowdria ruminantium were raised. Four mAbs of the IgG isotype reacted in western blots with a 32-kilodalton Cowdria protein (Cr32), which had previously been shown to be conserved and immunodominant. A fifth mAb of the IgM isotype recognized a 40-kDa Cowdria protein. The latter mAb was negative in an indirect fluorescent antibody test (IFA), whereas the other four were positive. mAb No. 4F10B4 showed the strongest signal in western blots using three different stocks of Cowdria. Immuno-gold labeling of Cowdria organisms in vitro using 4F10B4 showed that Cr32 has surface-exposed antigenic determinants. Using mAb 4F10B4, a competitive ELISA was developed which detected specific Cowdria antibodies in goat, sheep and cattle sera. Antibodies in animal sera competed with binding of mAb 4F10B4 to a crude sonicated Cowdria antigen obtained from infected endothelial cell cultures. The competition ELISA (CELISA) detected antibodies in 55 out of 70 (79%) goats experimentally infected with one of eight different Cowdria stocks. Fourteen out of the 15 sera which were shown negative in the CELISA were also negative in the IFA. Nevertheless, all 15 sera recognized some epitopes of the immunodominant Cowdria-specific 32 kDa protein as judged from their reaction with this protein in western blots. Overall, there was 89% agreement between CELISA and IFA considering all 70 goat sera. Moreover, antibodies were detected in nine out of nine sheep infected with one of three different stocks of Cowdria and in sera from calves experimentally infected by two different strains of heartwater. There were no cross-reactions with Ehrlichia phagocytophila antibodies in goat sera, nor with Anaplasma marginale antibodies in bovine sera. Lack of cross-reactivity and detection of antibodies to eight geographically widely distributed stocks of Cowdria, makes the competition ELISA a promising test for use in heartwater endemic areas.