Histopathological studies of experimental lyme disease in the dog.

Experimental borrelia infection was induced in 62 specific--pathogen-free beagle dogs by exposure to Ixodes scapularis ticks harbouring the spirochaete Borrelia burgdorferi. Clinical signs of Lyme disease occurred in 39/62 dogs, the remaining 23 being subclinically infected. Clinical signs consisted of one to six episodes of transitory lameness with joint swelling and pain, most commonly affecting the elbow or shoulder joints. The polymerase chain reaction and culture demonstrated that the dogs remained infected for up to 581 days. At necropsy, gross findings consisted of lymphadenopathy in the area of tick attachment. Microscopical changes consisted of effusive fibrinosuppurative inflammation or nonsuppurative inflammation, or both, affecting synovial membranes, joint capsules and associated tendon sheaths. Plasma cells dominated areas of chronic inflammation, with CD3(+) T cells being present in lesser numbers. Microscopical signs of arthritis were polyarticular and more widespread than indicated by clinical signs, and most of the subclinically affected animals also had synovitis. In areas of tick attachment to the skin, hyperkeratosis and a mixture of suppurative and nonsuppurative dermatitis were encountered. Lymphadenopathy in superficial lymph nodes resulted from follicular and parafollicular hyperplasia. In 14/62 dogs, lymphoplasmacytic periarteritis and perineuritis were noted, resembling lesions found in human Lyme disease and syphilis, in which an underlying microangiopathy has been proposed.

Protection against tick-transmitted Lyme disease in dogs vaccinated with a multiantigenic vaccine.

In an effort to develop a safe and effective vaccine for the prevention of Lyme borreliosis that addresses concerns raised over currently available vaccines, dogs were vaccinated twice with a multiantigenic preparation of Borrelia burgdorferi, strain N40, on days 0 and 20 of the experiment. About 70 and 154 days after the first immunization, dogs were challenged by exposing them to field-collected Ixodes scapularis ticks harboring B. burgdorferi. Vaccinated dogs were completely protected from infection by all criteria utilized to assess infection, developed high-titer anti-B. burgdorferi serum antibodies and growth inhibitory activity which persisted for over 200 days, and did not demonstrate any untoward consequence of vaccination. Serum absorption experiments revealed that borreliacidal and most likely protective antibodies in dogs receiving the multiantigenic preparation were not only elicited against the OspA antigen, but were also produced against additional yet to be determined targets on B. burgdorferi organisms. These data demonstrate that a multiantigenic vaccine is effective in preventing Lyme disease transmitted via the natural vector.

Evaluation of a commercial ELISA for diagnosis of paratuberculosis in cattle.

OBJECTIVE: To evaluate sensitivity and specificity of a new ELISA for antibodies against Mycobacterium avium subsp paratuberculosis. DESIGN: Cross-sectional observational survey. SAMPLE POPULATION: Serum samples from 590 cattle that were infected with M avium subsp paratuberculosis and 723 cattle that were not infected. PROCEDURE: Serum samples were tested by use of an ELISA for antibodies against M avium subsp paratuberculosis. RESULTS: Sensitivity of the test varied from 15.4 to 88.1%, depending on the clinical stage and bacterial shedding status of the cattle. CONCLUSIONS AND CLINICAL RELEVANCE: Results obtained with use of the new ELISA agreed favorably with those of a previous ELISA. Practitioners must be aware of variability in the sensitivity of the test, which depends on the clinical and shedding status of the cattle, because this may affect interpretation of test results.

Characterization of a Borrelia burgdorferi VlsE invariable region useful in canine Lyme disease serodiagnosis by enzyme-linked immunosorbent assay.

Sera collected from dogs experimentally infected with Borrelia burgdorferi by tick inoculation were analyzed for an antibody response to each of the six invariable regions (IRs; i.e., IR(1) to IR(6)) of VlsE, the variable surface antigen of B. burgdorferi. Six synthetic peptides (C(1) to C(6)), which reproduced the six IR sequences were used as peptide-based, enzyme-linked immunosorbent assay (ELISA) antigens. Two IRs, IR(2) and IR(6), were found to be immunodominant. Studies with serially collected serum samples from experimentally infected dogs revealed that the antibody response to IR(6) appears earlier and is stronger than that to IR(2). Thus, the IR(6) sequence alone appeared to be sufficient for serodiagnosis. When C(6) alone was used as antigen, the peptide-based ELISA was positive in 7 of 23 dogs (30%) as early as 3 weeks postinfection. All dogs (n = 33) became strongly positive 1 or 2 weeks later, and this response persisted for the entire study, which lasted for 69 weeks. Of 55 sera submitted by veterinarians from dogs suspected of having Lyme disease, 19 were also positive by the C(6) ELISA, compared to 20 positives detected by immunoblot analysis using cultured B. burgdorferi lysates as antigen. The sensitivity of using C(2) and C(6) together for detecting specific antibody in both experimentally infected and clinically diagnosed dogs was not better than sensitivity with C(6) alone, confirming that C(6) suffices as a diagnostic probe. Moreover, the C(6) ELISA yielded 100% specificity with serum samples collected from 70 healthy dogs, 14 dogs with infections other than B. burgdorferi, and 15 animals vaccinated with either outer surface protein A, whole-spirochete vaccines, or the common puppy-vaccines. Therefore, this C(6) ELISA was both sensitive and specific for the serodiagnosis of canine Lyme disease and could be used with vaccinated dogs.

High resolution refinement of beta-galactosidase in a new crystal form reveals multiple metal-binding sites and provides a structural basis for alpha-complementation.

The unrefined fold of Escherichia coli beta-galactosidase based on a monoclinic crystal form with four independent tetramers has been reported previously. Here, we describe a new, orthorhombic form with one tetramer per asymmetric unit that has permitted refinement of the structure at 1.7 A resolution. This high-resolution analysis has confirmed the original description of the structure and revealed new details. An essential magnesium ion, identified at the active site in the monoclinic crystals, is also seen in the orthorhombic form. Additional putative magnesium binding sites are also seen. Sodium ions are also known to affect catalysis, and five putative binding sites have been identified, one close to the active site. In a crevice on the protein surface, five linked five-membered solvent rings form a partial clathrate-like structure. Some other unusual aspects of the structure include seven apparent cis-peptide bonds, four of which are proline, and several internal salt-bridge networks. Deep solvent-filled channels and tunnels extend across the surface of the molecule and pass through the center of the tetramer. Because of these departures from a compact globular shape, the molecule is not well characterized by prior empirical relationships between the mass and surface area of proteins. The 50 or so residues at the amino terminus have a largely extended conformation and mostly lie across the surface of the protein. At the same time, however, segment 13-21 contributes to a subunit interface, and residues 29-33 pass through a "tunnel" formed by a domain interface. Taken together, the overall arrangement provides a structural basis for the phenomenon of alpha-complementation.

Structure and function of a human TAFII250 double bromodomain module.

TFIID is a large multiprotein complex that initiates assembly of the transcription machinery. It is unclear how TFIID recognizes promoters in vivo when templates are nucleosome-bound. Here, it is shown that TAFII250, the largest subunit of TFIID, contains two tandem bromodomain modules that bind selectively to multiply acetylated histone H4 peptides. The 2.1 angstrom crystal structure of the double bromodomain reveals two side-by-side, four-helix bundles with a highly polarized surface charge distribution. Each bundle contains an Nepsilon-acetyllysine binding pocket at its center, which results in a structure ideally suited for recognition of diacetylated histone H4 tails. Thus, TFIID may be targeted to specific chromatin-bound promoters and may play a role in chromatin recognition.

Status of Borrelia burgdorferi infection after antibiotic treatment and the effects of corticosteroids: An experimental study.

Sixteen specific-pathogen-free beagles were infected with Borrelia burgdorferi. Three groups of 4 dogs were treated with antibiotics for 30 consecutive days starting 120 days after tick exposure; 4 dogs were untreated controls. At day 420 after tick exposure and again before euthanasia, 2 dogs of each group were treated with prednisone for 14 days. All dogs contracted infection and 11 developed acute arthritis 50-120 days after exposure. After day 120, one of 12 antibiotic-treated dogs and 2 of 4 untreated dogs became lame. Antibiotic therapy reduced the frequency of Borrelia-positivity in subsequent skin biopsy samples. After prednisone treatment, both control dogs developed severe polyarthritis. At euthanasia, single tissues of the antibiotic-treated dogs and multiple tissues of all control dogs were Borrelia-positive by polymerase chain reaction. Viable spirochetes were not recovered from antibiotic-treated dogs. Two antibiotic-treated dogs showed histologic evidence of minimal lesions, whereas all control dogs had mild polyarthritis with periarteritis.

Cell mediated and humoral immune responses of white-tailed deer experimentally infected with Mycobacterium bovis.

The objective of this study was to improve the understanding of immune responses of whitetailed deer (Odocoileus virginianus) infected with Mycobacterium bovis. Ten mature, female, white-tailed deer were inoculated by intratonsilar instillation of 2 x 10(3)or 2 x 10(5)colony-forming units of M. bovis. Lymphocyte proliferation and humoral response to M. bovis PPD and the M. bovis protein, MPB70 were measured. Deer were tested for exposure to M. bovis by the comparative cervical skin test. Biopsy specimens of skin test sites were examined microscopically and immunohistochemically. The comparative cervical skin test correctly identified all M. bovis -inoculated deer as exposed to M. bovis. Lymphocyte proliferative responses to MPB70 were more consistent than responses to M. bovisPPD in M. bovis -inoculated deer. Antibody responses were more prominent in deer with disseminated disease than in deer with localised disease. The cellular components of delayed-type hypersensitivity reactions at skin test sites were similar to tuberculin reactions in other species. T lymphocytes of the gamma/delta phenotype were seen in increased numbers in M. bovisPPD injection sites.

Experimental infection of ponies with Borrelia burgdorferi by exposure to Ixodid ticks.

Seven specific-pathogen-free (SPF) ponies, 1-5 years old, were exposed to Borrelia burgdorferi-infected adult ticks while being treated with dexamethasone over 5 consecutive days. One SPF pony (pony No. 178) was first exposed to laboratory-reared nymphs without B. burgdorferi infection and 3 weeks later was exposed to B. burgdorferi-infected adult ticks with concurrent dexamethasone treatment for 5 consecutive days. Four uninfected ponies treated with dexamethasone, exposed to laboratory-reared ticks without B. burgdorferi infection served as uninfected controls. Clinical signs, bacteriologic culture, polymerase chain reaction (PCR) for bacterial DNA, immunologic responses, and gross lesions and histopathologic changes were investigated during the experiment or at necropsy 9 months after tick exposure. In all of the seven challenged ponies, infection with B. burgdorferi was detected from monthly skin biopsies and various tissues at postmortem examination by culture and by PCR. However, pony No. 178 exposed to laboratory-reared nymphs (without B. burgdorferi infection) and challenged with B. burgdorferi-infected adult ticks 2 months later did not develop a B. burgdorferi infection. All of the infected ponies seroconverted. Control ponies and pony No. 178 were negative by culture, PCR, and serology. Except for skin lesions, we failed to induce any significant histopathologic changes in this study. This is the first report of successful tick-induced experimental infection in ponies by exposure to B. burgdorferi-infected ticks. This Lyme disease model will be very useful to evaluate efficacy of vaccines against the Lyme agent and the effect of antibiotic therapy on horses infected with B. burgdorferi.

Efficacy of a nonadjuvanted, outer surface protein A, recombinant vaccine in dogs after challenge by ticks naturally infected with Borrelia burgdorferi.

In a blinded, controlled study, thirty purpose-bred, Borrelia burgdorferi negative, mixed-breed dogs 10 to 12 weeks of age were randomly divided into three groups of ten animals each for the purpose of evaluating a recombinant nonadjuvanted B. burgdorferi OspA vaccine (Recombitek Lyme [Merial Limited]) for efficacy and safety. Two groups received two doses of two different lots ofa nonadjuvanted, OspA, recombinant vaccine; the third group served as nonvaccinated controls. All dogs were challenged 3 weeks after the second vaccination with blacklegged deer ticks (Ixodes scapularis) harvested from a B. burgdorferi endemic area in Rhode Island. Clinical signs, antibody titers by ELISA, Western blot assays, and isolation and polymerase chain reaction analyses of spirochetes from biopsy specimens were used to evaluate vaccine efficacy. Direct fluorescent antibody assay was used to evaluate the infection rate in the challenge ticks and in naïve ticks allowed to feed on study dogs after the dogs were infected (xenodiagnosis). Vaccinates responded with high levels of antibodies (determined by ELISA and measured by optical density [OD]), which did not rise after challenge. Vaccinates demonstrated no clinical signs, negative isolation of spirochetes on biopsy, only an OspA antibody pattern on Western blot assay, and negative isolation of spirochetes on biopsy, confirming that the vaccine blocked infection with B. burgdorferi in all vaccinated dogs (20/20). Control dogs demonstrated clinical signs (2/10), antibodies characteristic of infection with B. burgdoferi (10/10), isolation of spirochetes (10/10), and polymerase chain reaction (PCR) detection of spirochetes (9/10). The recombinant, nonadjuvanted B. burgdoferi vaccine protected 100% of vaccinates against infection after a severe challenge that infected 100% of control dogs. The OspA vaccine proved to be highly safe and effective in this study.

Vaccination against lyme disease with recombinant Borrelia burgdorferi outer-surface protein A (rOspA) in horses.

Eight 1-year-old ponies were vaccinated with recombinant OspA (ospA gene derived from B. burgdorferi B31) with adjuvant (aluminium hydroxide). Four ponies were used as non-vaccinated controls with adjuvant. One hundred and twelve days after the first vaccination, the vaccinated and non-vaccinated ponies were challenged by exposure to B. burgdorferi-infected adults tick (Ixodes scapularis) collected from Westchester County, New York (tick infection rate >/=60%). Protection from infection was evaluated by culture for B. burgdorferi from three monthly skin biopsies taken near the site of tick bites. B. burgdorferi was not isolated from any of the vaccinated ponies. In contrast, three of four control ponies challenged by tick exposure were skin culture positive. At the time of tick exposure, vaccinated ponies had antibody to B. burgdorferi demonstrable by KELA (kinetic-ELISA), western blot and a serum growth inhibition assay. Antibodies in the challenge control ponies were only detectable by two to three months after tick exposure and remained at intermediate levels until termination of the study. By western blot analysis, antibodies to OspA first appeared in the sera of vaccinated ponies three weeks after the first vaccination. The absence of additional bands, known to develop when the animal is infected, suggests that infection was blocked after tick exposure of vaccinated ponies. Results from this study show that vaccination with recombinant OspA protected ponies against infection after experimental challenge with B. burgdorferi-infected ticks.

Serological discrimination of dogs infected with gastric Helicobacter spp. and uninfected dogs.

Characterization of the humoral immune responses of people to Helicobacter pylori infection has facilitated the investigation of the host response to bacterial virulence factors and the development of sensitive and specific diagnostic tests. Dogs are commonly infected with gastric Helicobacter spp., but the presence of multiple Helicobacter spp. and possible coinfection in individual dogs have complicated serological evaluation. Evaluation of the antigenic homology of Helicobacter spp. revealed that the major protein bands of Helicobacter felis and Helicobacter bizzozeronii, two Helicobacter spp. that infect dogs, were very similar to UreA (29 to 31 kDa), UreB (63 to 66 kDa), and HSP (58 to 60 kDa) of H. pylori, and sera from infected and uninfected dogs bound in a similar way to each antigen. Immunoblotting and an enzyme-linked immunosorbent assay (ELISA) with H. felis ATCC 49179 antigen were performed with 101 serum samples (from 78 infected dogs and 23 uninfected dogs). Samples from uninfected dogs (median = 8) had fewer bands on immunoblotting than samples from infected dogs (median = 16) (P < 0.05). Combinations of the presence of any two of the low-molecular-mass bands (19, 25, 30, 32, and 37 kDa) or the high-molecular-mass bands (86 and 94 kDa) were found almost solely in samples from infected dogs (P < 0.0001). Kinetic ELISA results were significantly higher for samples from infected dogs (median = 0. 0802 optical density unit [OD]/min) than for samples from uninfected dogs (median = 0.01428 OD/min). The combination of ELISA and immunoblotting results gave a specificity of 95.6% and a sensitivity of 79.8%. No correlation between ELISA results, colonization density, degree of inflammation, and presence of lymphoid follicles was observed. The results indicate substantial antigenic homology between H. felis, H. pylori, and H. bizzozeronii. The combination of ELISA and immunoblotting was a highly specific and moderately sensitive indicator of infection. The degree of seropositivity assessed by ELISA was not related to bacterial colonization density, the degree of gastric inflammation, or the presence of lymphoid follicles.

Validation of serological assays for diagnosis of infectious diseases.

Assay validation is a series of the following interrelated processes: an experimental process: reagents and protocols are optimised by experimentation to detect the analyte with accuracy and precision, and to ensure repeatability and reproducibility in the assay. a relative process: its diagnostic sensitivity and diagnostic specificity are calculated relative to test results obtained from reference animal populations of known infection/exposure status. a conditional process: classification of animals in the target population as infected or uninfected is conditional upon how well the reference animal population used to validate the assay represents the population to which the assay will be applied (accurate predictions of the infection status of animals from test results and predictive values of positive and negative test results are conditional upon the estimated prevalence of disease/infection in the target population) an incremental process: confidence in the validity of an assay increases over time when use confirms that it is robust as demonstrated by accurate and precise results (the assay may also achieve increasing levels of validity as it is upgraded and extended by adding reference populations of known infection status) a continuous process: the assay remains valid only insofar as the assay continues to provide accurate and precise results as proved through statistical verification. Therefore, validation of diagnostic assays for infectious diseases does not end with a time-limited series of experiments based on a few reference samples. Rather, it is a process that also requires constant vigilance and maintenance, along with reassessment of its performance characteristics for each population of animals to which it is applied. It is certain that the current movement to develop and implement accreditation criteria for veterinary diagnostic laboratories may be of little worth unless there is some assurance that the assays conducted in such laboratories are properly validated. Fully accredited laboratories may generate highly reproducible test results, but the results may still misclassify animals as to their infection status due to an improper assay validation process. Therefore, assay validation is foundational to the core product of veterinary diagnostic laboratories--test results and their interpretation.

Interpretations of antibody responses to Salmonella enterica serotype enteritidis gm flagellin in poultry flocks are enhanced by a kinetics-based enzyme-linked immunosorbent assay.

Many regulatory and diagnostic programs for the detection of Salmonella enterica serotype Enteritidis infection in commercial poultry flocks have relied on rapid Pullorum agglutination tests to screen birds because of the shared antigens of S. enterica Enteritidis and S. enterica Pullorum and Gallinarum; however, the use of the enzyme-linked immunosorbent assay (ELISA) format affords better analytical sensitivity than crude agglutination tests. In this study, we adapted our earlier conventional indirect ELISA, using gm flagellin as the antigen, to a kinetics-based, computer-controlled ELISA (KELA). The KELA was used to screen for flagellin antibody from three commercial flocks: (i) a large flock involved in a U.S. Department of Agriculture trace back from a human S. enterica Enteritidis foodborne outbreak (n = 3,209), (ii) a flock infected with the endemic S. enterica Enteritidis serotype but which also had multiple other Salmonella serotypes (n = 65), and (iii) an S. enterica Pullorum-infected flock (n = 12). The first flock (S. enterica Enteritidis prevalence of 2.45% based on culture) provided a field test of the KELA and allowed the calculation of diagnostic sensitivity (D-Sn) and diagnostic specificity (D-Sp). With a cutoff of 10 (used for screening flocks [i.e., high sensitivity]), the KELA has a D-Sn of 95.2% and a D-Sp of 18.5%; with a cutoff of 140 (used in confirmatory flock testing [i.e., high specificity]), the KELA has a D-Sn of 28.0% and a D-Sp of 99.1%. We found that with a cutoff of 60 (D-Sn = 63.1%; D-Sp = 91.6%), we could eliminate reactions in the KELA caused by other non-S. enterica Enteritidis salmonellae. The KELA was also compared to two commercial rapid Pullorum tests, the Solvay (D-Sn = 94.9%; D-Sp = 55.5%) and the Vineland (D-Sn = 62.0%; D-Sp = 75.3%).

Clinical manifestations, pathogenesis, and effect of antibiotic treatment on Lyme borreliosis in dogs.

BACKGROUND: Borrelia burgdorferi, the causative agent of Lyme disease, infects humans and animals. In humans, the disease primarily affects the skin, large joints, and the nervous system days to months after infection. Data generated with appropriate animal model help to understand the fundamental mechanisms of the disease. OBJECTIVE: 1) More clearly define the clinical manifestation and pathogenetic mechanisms of Lyme disease in dogs; 2) evaluate the effect of antibiotics in dogs infected with B. burgdorferi; 3) describe the effects of corticosteroids on dogs persistently infected with B. burgdorferi. DESIGN: Specific-pathogen-free beagles were infected with B. burgdorferi using ticks collected in an endemic Lyme disease area. Clinical signs were recorded daily. Antibody titers were measured by ELISA at two-week intervals. B. burgdorferi organisms were detected in tissues by culture and PCR. Synovial fluids were evaluated microscopically and with a chemotaxis cell migration assay. Histological sections were examined for pathological lesions. Specific cytokine up-regulation in tissues was detected by RT-PCR. INTERVENTIONS: In three separate experiments, B. burgdorferi-infected dogs received antibiotic treatment (amoxicillin; azithromycin; ceftriaxone; doxycycline) for 30 consecutive days. Two subclinical persistently infected dogs received oral prednisone for 14 consecutive days starting at day 420 post-infection. RESULTS: Dogs developed acute arthritis in the joints closest to the tick bites after a median incubation period of 68 days. Synovial membranes of lame and non-lame dogs produced the chemokine IL-8 in response to B. burgdorferi. Antibiotic treatment prevented or resolved episodes of acute arthritis, but failed to eliminate the bacterium from infected dogs. Corticosteroid treatment reactivated Lyme disease in persistently infected dogs, which had not received antibiotics previously. CONCLUSIONS: B. burgdorferi disseminates through tissue by migration following tick inoculation, produces episodes of acute arthritis, and establishes persistent infection. The spirochete survives antibiotic treatment and disease can be reactivated in immunosuppressed animals.

Transcription properties of a cell type-specific TATA-binding protein, TRF.

Eukaryotic cells are thought to contain a single TATA-binding protein (TBP) that directs transcription by cellular RNA polymerases. Here we report a cell type-specific TBP-related factor (TRF) that can form a stable TRF/IIA/IIB TATA DNA complex and substitute for TBP in directing RNA polymerase II transcription in vitro. Transfection studies reveal that TRF can differentially mediate activation by some enhancer proteins but not others. Like TBP, TRF forms a stable complex containing multiple novel subunits, nTAFs. Antibody staining of embryos and polytene chromosomes reveals cell type-specific expression and gene-selective properties consistent with the shaker/male sterile phenotype of trf mutants. These findings suggest TRF is a homolog of TBP that functions to direct tissue- and gene-specific transcription.

Borrelia burgdorferi migrates into joint capsules and causes an up-regulation of interleukin-8 in synovial membranes of dogs experimentally infected with ticks.

Twenty 6-week-old specific-pathogen-free beagles were infected with Borrelia burgdorferi by tick challenge, and five uninfected dogs served as controls. During the study, all dogs were monitored for infection, clinical signs, and antibody response against B. burgdorferi. During episodes of lameness or postmortem, synovial fluids from each dog were examined for volume, cell number, polymorphonuclear leukocyte (PMN) content, cell viability, and chemotactic activity. Twenty-five tissues collected postmortem from each dog were tested for interleukin-8 (IL-8) mRNA, tumor necrosis factor alpha (TNF-alpha) mRNA, presence of live spirochetes, and histopathological changes. Thirteen infected dogs (group A), which seroconverted rapidly (maximum titers within 50 to 90 days), developed acute and severe mono- or oligoarthritis almost exclusively in the limb closest to the tick bite (median incubation period, 66 days). Synovial fluids of the arthritic joints collected during episodes of lameness had significantly elevated volume, cell count, PMN proportion, cell viability, and chemotactic activity for PMNs. The remaining joints of the same animals contained synovial fluids with elevated chemotactic activity and cell viability. Twelve dogs tested positive for IL-8 mRNA in multiple tissues (synovia, pericardium, and peritoneum), and 10 dogs expressed TNF-alpha mRNA, but only in the tributary lymph nodes of the inflamed joints. Histological examinations revealed severe poly- or oligoarthritis and moderate to severe cortical hyperplasia in draining lymph nodes of the inflamed joints in all 13 dogs. Seven infected dogs with mild or no clinical signs (group B) seroconverted slowly (peak titers after 90 days), and only some joint fluids showed chemotactic activity, which on average was lower than that in inflamed and noninflamed joints from dogs in group A. Four dogs expressed IL-8 mRNA (in the synovia and pericardium), and three dogs had TNF-alpha mRNA in tributary lymph nodes. Histologically, nonsuppurative arthritis was found in multiple joints, and mild to moderate cortical hyperplasia was found in draining lymph nodes. Five uninfected dogs without lameness (group C) had normal synovial fluids and tissues. In all infected dogs, live spirochetes were demonstrated more frequently in tissues of the somatic quadrant closest to the tick bite than in tissues further from the site of infection, suggesting that dissemination of B. burgdorferi occurs more by migration than by blood-borne spread. From these studies employing a canine model of B. burgdorferi infection, we conclude that IL-8 is involved in the pathogenesis of acute Lyme arthritis.

Assessing the validity of serodiagnostic test results.

When questionable serological results have been obtained from testing laboratories or test kits used in the clinic, the veterinarian either can accept the results at face value, and suffer the probable consequences of misdiagnoses, or take steps to ascertain if indeed the assay was properly validated and the result properly interpreted. It should not be assumed that assays offered by all commercial testing laboratories are fully validated. Neither should it be assumed that test kits, because they are licensed by the USDA, will give accurate answers to all serological questions. When evidence suggests that an assay was fully validated, test results produced by that assay still must be interpreted by the veterinarian within the context of the probability of disease for the patient or groups of animals before testing, or misclassification of infection/disease status of patients may occur. The astute veterinarian will take steps to clarify if the testing laboratory or the test kits being used are performing adequately. Additionally, all test results in that practice will be interpreted in the context of their predictive values. Simplified methods for accomplishing these goals in a timely manner have been provided and will lead to more reasoned clinical diagnoses.

Lyme disease: laboratory diagnosis of infected and vaccinated symptomatic dogs.

Serological assays for detection of canine antibodies to the Lyme agent generally have been difficult to validate because an acceptable standard of comparison such as unequivocal proof of infection status has not been available. For practical and logistical reasons, it has not been possible to use culture of organism from infected animals, seroconversion in a large number of field dogs, or clinical criteria as the standard of comparison for validation of assays. Therefore, estimates of diagnostic sensitivity and specificity based on an appropriate gold standard have not been available. When it was discovered how to infect laboratory dogs via ticks infected with Borrelia burgdorferi, it was possible to define the kinetics and magnitude of the antibody response that might be expected in nature. ELISA and Western immunoblot data from experimental dogs were then compared and correlated with results of the same tests on dogs from endemic and nonendomic areas. Coupled with studies on cross-reactive antibodies elicited from other infectious agents or autoimmune phenomena, it was possible to account for interfering antibodies and to establish estimates of diagnostic sensitivity and specificity for the ELISA based on objective criteria. Such validated assays can predict, with a relatively high degree of proficiency, the infection and/or vaccinal status of animals. These assays have shown that some dogs, vaccinated with the commercially available whole-cell Lyme bacterins develop typical signs of Lyme disease but have no evidence of an underlying infection; antibody elicited only by the vaccine and not by infection is detectable in these animals. Western immunoblot can also confirm infection in animals of equivocal ELISA status if their bands have been evaluated for specificity of antibodies to B burgdorferi. Serology can be a very useful aid in the diagnosis of Lyme disease, but it requires that the assays used have been subjected to rigorous validation criteria. When that is not performed, an unacceptable level of false-positive and false-negative test results is virtually assured.