Validation of an in-clinic enzyme-linked immunosorbent assay kit for diagnosis of Borrelia burgdorferi infection in horses.

Confirmation of Borrelia burgdorferi infection in horses has required enzyme-linked immunosorbent assay (ELISA) or Western blot tests performed by reference laboratories. An in-clinic C6 ELISA SNAP kit has been marketed for dogs. This canine kit was evaluated for horses using serum from experimentally infected ponies. Serum samples originated from 2 previous studies. In the first study, 7 ponies were exposed to B. burgdorferi-infected ticks; 4 ponies served as uninfected controls. Serum samples were obtained bimonthly for 9 months. In the second study, 16 ponies were exposed to B. burgdorferi-infected ticks. After confirmation of infection by skin culture, polymerase chain reaction (PCR), and serology, the ponies were allocated to 4 groups that received tetracycline, doxycycline, ceftiofur, or no treatment. Serum samples were obtained monthly, both before and after antibiotic treatments, for 11 months. For the current study, selected samples (n = 220) from both studies were tested with IDEXX SNAP Heartworm Ab/Borrelia burgdorferi Ab/Ehrlichia canis Ab Test Kits. Tested samples included samples taken before infection, from various times postinfection, and after antibiotic treatments. Results from confirmed positive or negative samples were used to determine sensitivity and specificity of the assay. Results indicate that the test kits have fair sensitivity (63%) and very high specificity (100%) for horses recently infected with B. burgdorferi. Validation of this test provides equine practitioners with an inexpensive, in-clinic method to confirm infection, although its moderate sensitivity may result in a moderate chance of a false negative test.     

Experimental induction of chronic borreliosis in adult dogs exposed to Borrelia burgdorferi-infected ticks and treated with dexamethasone.

OBJECTIVE: To develop a method to experimentally induce Borrelia burgdorferi infection in young adult dogs. ANIMALS: 22 healthy Beagles. PROCEDURE: All dogs were verified to be free of borreliosis. Twenty 6-month-old dogs were exposed to Borrelia burgdorferi-infected adult ticks and treated with dexamethasone for 5 consecutive days. Two dogs not exposed to ticks were treated with dexamethasone and served as negative-control dogs. Clinical signs, results of microbial culture and polymerase chain reaction (PCR) testing, immunologic responses, and gross and histologic lesions were evaluated 9 months after tick exposure. RESULTS: Predominant clinical signs were episodic pyrexia and lameness in 12 of 20 dogs. Infection with B burgdorferi was detected in microbial cultures of skin biopsy specimens and various tissues obtained during necropsy in 19 of 20 dogs and in all 20 dogs by use of a PCR assay. All 20 exposed dogs seroconverted and developed chronic nonsuppurative arthritis. Three dogs also developed mild focal meningitis, 1 dog developed mild focal encephalitis, and 18 dogs developed perineuritis or rare neuritis. Control dogs were seronegative, had negative results for microbial culture and PCR testing, and did not develop lesions. CONCLUSIONS AND CLINICAL RELEVANCE: Use of this technique successfully induced borreliosis in young dogs. Dogs with experimentally induced borreliosis may be useful in evaluating vaccines, chemotherapeutic agents, and the pathogenesis of borreliosis-induced arthritis.     

Synovial lesions in experimental canine Lyme borreliosis

Borrelia burgdorferi is the causative agent of Lyme disease, which is mainly characterized by lameness in dogs. More than 95% of naturally infected dogs are asymptomatic or subclinical; however, in experimental studies, histologic synovial lesions are consistently observed in asymptomatic dogs inoculated with B. burdgorferi. This study investigates the ability of a synovial histopathologic scoring system, clinicopathologic data, and polymerase chain reaction (PCR) testing to differentiate between B. burgdorferi-infected and uninfected dogs. Eighteen 18-week-old beagles were subject to challenge with B. burgdorferi-infected wild-caught ticks (Ixodes scapularis), and 4 uninfected dogs served as controls. Infection was confirmed by serology (ELISA) and PCR amplification of B. burgdorferi-specific DNA of skin biopsies taken at the tick attachment site. A synovial scoring system from human medicine was adapted and implemented on postmortem synovial samples to discriminate infected and noninfected animals. Application of this system to elbows and stifles with a cumulative joint score cutoff > 4 showed a sensitivity of 88.2% and a specificity of 100%, with a positive likelihood ratio of infinity and a negative likelihood ratio of 0.12. Complete blood count, serum biochemistry, urinalysis, urine protein:creatinine, urine PCR, synovial and lymph node cytology, and synovial PCR were evaluated but were not reliable indicators of clinical disease.     

Borrelia burgdorferi sensu lato and Rickettsia spp. infections in hard ticks (Ixodes ricinus) in the region of Hanover (Germany)

In a total of 605 Ixodes (I.) ricinus ticks collected in the spring-months March, April and May 2005, quantitative real time PCR (qPCR) revealed 26.6% Borrelia (B.) burgdorferi sensu lato (sl)-positive ticks, i. e. divided by sex and stage into 31.9% positive adults (34.8% females and 29.0% males) and 18.5% positive nymphs. Mono-infections with genospecies from the B. burgdorferi sl-complex were found in over two thirds of the positive individuals, whereas almost one third showed double- or even triple-infections. Genospecies-specific conventional PCR determined B. afzelii as the most frequent genospecies followed by B. garinii, B. spielmanii, B. valaisiana and B. burgdorferi sensu stricto (ss). Rickettsia spp. were found in 34.2% of the collected ticks, divided into 37.6% adults (42.5% females and 32.8% males) and 29.0% nymphs. Co-infections of Rickettsia-positive ticks with B. burgdorferi sl spirochaetes were present in 10.1% of the ticks. Thereby, adult ticks exhibited a co-infection rate of 13.4% (15.5% females and 11.3% males) and nymphs of 5.0%. Independently of the above mentioned study, 3939 Ixodes ticks, sent in between 2006 and 2010 for B. burgdorferi sl-diagnostic, were examined by qPCR exclusively for B. burgdorferi sl. The resulting B. burgdorferi sl prevalence was 23.1% and 24.4% in 2006 and 2007, respectively, followed by a continuous decrease to 12.8% in 2010. To analyse whether this observed decrease in infection frequency is due to sampling bias, in a current study randomly sampled ticks collected from defined sites equally distributed over the city of Hanover are investigated in a statistically relevant sample size.     

Comparison of direct fluorescent antibody staining and real-time polymerase chain reaction for the detection of Borrelia burgdorferi in Ixodes scapularis ticks.

Borrelia burgdorferi, the agent responsible for causing Lyme disease in humans and animals, is transmitted via the bite of infected Ixodes spp. ticks. Ticks removed from humans and animals are routinely tested by diagnostic laboratories to determine if they are infected with these bacteria. The objective of this study was to compare the efficacy of 2 commonly used methods, direct fluorescent antibody staining and real-time polymerase chain reaction (PCR), for the detection of B. burgdorferi in Ixodes scapularis ticks. One hundred and twenty-seven adult I. scapularis ticks collected in Connecticut, a Lyme disease endemic area, were tested, and results were compared. Results showed 24.8% ticks tested positive for Borrelia spp. by fluorescent antibody testing and 32.5% ticks were positive for B. burgdorferi by real-time PCR testing. When ticks were grouped into categories by level of engorgement (unengorged, partially engorged, and fully engorged), 95% of unengorged ticks, 90.5% of partially engorged, and 86.8% of engorged ticks tested were in agreement. Ten of the 127 ticks examined were too dehydrated to be tested by the fluorescent antibody technique; half of these tested positive by PCR. Real-time PCR appears to be the better of these 2 methods for the diagnosis of this bacterial infection in I. scapularis ticks.     

The ability of a topical novel combination of fipronil, amitraz and (S)-methoprene to protect dogs from Borrelia burgdorferi and Anaplasma phagocytophilum infections transmitted by Ixodes scapularis

Healthy, purpose-bred laboratory beagle dogs that had not been exposed to ticks and were seronegative for Borrelia burgdorferi and Anaplasma phagocytophilum were randomly assigned to four groups of eight dogs each. Control group 1 was not treated. Groups 2, 3 and 4 were treated with a single topical application of a new formulation of fipronil, amitraz and (S)-methoprene (CERTIFECT?, Merial Limited, GA, USA) at 28, 21 or 14 days prior to tick infestation, respectively. Each dog was infested with 25 female and 25 male field-collected adult Ixodes scapularis ticks that had infection rates of 66% for B. burgdorferi sensu stricto and 23% for A. phagocytophilum, as determined by polymerase chain reaction. Two and five days after tick infestation, control dogs had an average of 9.5 and 13.9 attached adult female ticks, respectively, whilst the 24 treated dogs remained tick-free aside from a single tick on the 2nd day after infestation. Serial serological tests demonstrated that the ticks successfully infected 8/8 control dogs with B. burgdorferi and co-infected 6/8 with A. phagocytophilum. B. burgdorferi infection also was confirmed in most control dogs by culture (6/8) and PCR (7/8) of skin biopsies. In contrast, CERTIFECT protected all 24 treated dogs against infection by both B. burgdorferi and A. phagocytophilum, as demonstrated by their negative serological tests throughout the study and the absence of any positive skin biopsy culture or PCR in these dogs.     

Antibiotic treatment of experimentally Borrelia burgdorferi-infected ponies

The objective of this study is to determine whether doxycycline, ceftiofur or tetracycline could be effectively used to treat equine Lyme disease. Ponies experimentally infected with Borrelia burgdorferi by tick exposure were treated with doxycycline, ceftiofur or tetracycline for 4 weeks (28 days). Doxycyline and ceftiofur treatment were inconsistent in eliminating persistent infection in this experimental model. However, tetracycline treatment seems to eliminate persistent infection. Although serum antibody levels to B. burgdorferi in all ponies declined gradually after antibiotic treatment, three out of four ponies treated with doxycline and two out of four ponies treated with ceftiofur, serum KELA titers were raised again 3 month after treatment was discontinued. Five months after antibiotic treatment, tissues aseptically collected at necropsy from ponies with increased antibody levels after antibiotic treatment also showed culture positive to B. burgdorferi in various post-mortem tissues. However, all four-tetracycline treatment ponies showed a negative antibody level and culture negative from post-mortem tissues. Untreated infected ponies maintained high KELA titers throughout the study and were tissue culture positive.     

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.     

Transmission of Anaplasma phagocytophila (human granulocytic ehrlichiosis agent) in horses using experimentally infected ticks (Ixodes scapularis)

Most human granulocytic ehrlichiosis (HGE) studies carried out in horses use needle inoculation of infected leucocytes or cell cultures. This route of inoculation does not accurately reflect natural infection of the tick-borne agent. To investigate whether tick transmission influences the course of granulocytic ehrlichiosis in the horse model, experimental transmission through infected laboratory-reared Ixodes scapularis ticks was attempted into two healthy horses. One additional horse served as negative control and was exposed to uninfected ticks. Eleven days after exposure to nymphal or adult ticks infected with Anaplasma phagocytophila (HGE agent) the two horses developed severe clinical and laboratory signs consistent with granulocytic ehrlichiosis. Bacteraemia was determined at various time points in the two horses by observation of morulae within neutrophils and by detection of A. phagocytophila genomic DNA by PCR of peripheral blood leucocytes. Further, both horses seroconverted. In contrast the control horse stayed uninfected. The results demonstrate that A. phagocytophila can be experimentally transmitted by infected nymphal and adult ticks and that the agent is able to produce a severe disease, similar to naturally occurring cases. Therefore, tick transmission is highly reproducible and can be successfully used in the equine animal model in order to study HGE.     

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.     

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.     

Up-regulation of inducible nitric oxide synthase mRNA in dogs experimentally infected with Borrelia burgdorferi

The up-regulation of the inducible nitric oxide synthase (iNOS) mRNA was determined by RT-PCR in 25 tissues each from 22 specific pathogen-free (SPF) dogs experimentally infected with Borrelia burgdorferi by tick exposure and from five uninfected control dogs. Using primers specific for a homologous region of the human and canine iNOS sequence, and canine macrophage mRNA, we isolated and partially sequenced canine iNOS. A sequence of 1775 bases was obtained and primers specific for canine iNOS mRNA constructed to investigate the expression of iNOS in dog tissues in response to infection with B. burgdorferi. In 12 out of 22 dogs infected with B. burgdorferi, acute lameness occurred within 55-82 days after infection whereas the other 10 dogs showed no or only mild clinical signs despite persistent infection up to Day 175. The numbers of iNOS mRNA-positive tissues in dogs with acute lameness were significantly higher than in dogs without lameness, while uninfected dogs showed only negligible iNOS expression. Dogs with acute lameness also had higher numbers of borrelia-positive tissues as well as higher scores in histopathological evaluations than infected dogs without lameness. Our results show that the expression of iNOS mRNA is related to the number of B. burgdorferi-positive tissues and the severity of inflammation as assessed by histopathology. These results implicate an up-regulation of the iNOS mRNA as part of the host's immune response to borrelia infection and a possible role for NO in the pathogenesis of canine Lyme arthritis.     

Acute experimentally induced aflatoxicosis in the weanling pony

Nineteen weanling ponies and 1 adult pony were given a single oral dose of aflatoxin B1 (AFB1). Dosages were: 0, 0.5, 1, 2, 4, 5, 6, and 7.4 mg of AFB1/kg of body weight. Vital signs were monitored, and whole blood and serum collected for analysis of serum enzymes, prothrombin time, blood cell counts, and serum urea nitrogen. Ponies that died were examined for gross lesions, and tissues were collected for histopathologic examination and analysis of AFB1 and AFM1 residues. Two of the 4 ponies given the 2 mg/kg dose and all ponies given the larger dosages died within 76 hours. Clinical signs included increased rectal temperature, faster heart and respiratory rates, abdominal straining, bloody feces, and tetanic convulsions. At necropsy, ponies that died of acute aflatoxicosis showed visceral petechiae and hepatic focal lesions. Histopathologic changes included severe hepatic necrosis, vacuolation, and bile duct hyperplasia. Aflatoxins B1 and M1 were recovered from liver, kidney, skeletal muscle, and gastrointestinal contents. One other pony given the 2 mg/kg dose died 32 days after dosing, and 1 control pony died after 70 days. Continuous elevations in prothrombin time and serum aspartate aminotransferase, alanine aminotransferase, and gamma-glutamyl transpeptidase levels were observed in ponies dosed at 4 mg/kg or more. Significant (P less than 0.05) elevations in these values, which peaked 2 to 3 days after dosing, were seen in ponies given the 2 mg/kg dose. This group also had significant increases over controls in PCV and hemoglobin concentration 5 days after dosing.     

Larval Gulf Coast ticks (Amblyomma maculatum) [Acari: Ixodidae] as host for Hepatozoon americanum [Apicomplexa: Adeleorina].

Laboratory-reared larval Gulf Coast ticks (GCTs) (Amblyomma maculatum) were exposed experimentally and found to acquire Hepatozoon americanum infection while feeding on parasitemic dogs. These ticks supported gamogonic and sporogonic development of the apicomplexan, and oocysts from newly molted nymphs were infectious for a dog. Other nymphs from this cohort that were allowed to feed on a blood-parasite naive sheep molted normally; the resulting adult ticks contained oocysts that were infectious for another dog. Merogonic development of H. americanum in the dogs and the resulting lesions/disease appeared similar, irrespective of whether infectious oocysts were derived from nymphal or adult ticks that acquired infection as larvae. In the system previously known, nymphal ticks acquire infection and adults harbor infective oocysts, which vertebrate hosts ingest. Given that larval A. maculatum can acquire infection and nymphs can harbor viable oocysts as demonstrated by this study, the potential variety of vertebrate hosts that can alternate with GCTs in maintaining an endemic cycle is considerably expanded.     

Estimating Lyme disease risk using pet dogs as sentinels

The reported number of cases of Lyme disease, Borrelia burgdorferi sensu lato, is thought to have increased in the UK over the past decade, but consistent surveillance data are lacking. Here the prevalence of B. burgdorferi in ticks attached to pet dogs was examined - using them as sentinels for human disease risk. Dogs give a good indication of the exposure of their human owners to infected ticks, since they largely share the same environment and visit the same outdoor areas. PCR was used to test 739 tick samples collected from 3534 dogs selected at random as they visited veterinary practices over a period of six months. Overall, the prevalence of infected ticks on all dogs was 0.5% giving an estimated 481 infected ticks per 100,000 dogs. The data suggest that the prevalence of Borrelia in the UK tick population is considerably higher than most recent estimates indicate.     

Specific serum protein changes associated with primary and secondary Strongylus vulgaris infections in pony yearlings

The concentrations of haptoglobin, immunoglobin (Ig)G(T) and IgG were measured in the serum of four previously parasite-free pony yearlings following a single dose of 700 (Group H) or 200 (Group L) stage three Strongylus vulgaris larvae (L3) and following a reinfection with the same doses 34 weeks later. The results are compared with an uninfected control pony. The haptoglobin concentration increased during Weeks 1 to 6 and 14 to 17 after infection in the serum of the ponies receiving 200 L3, but in only one pony dosed with 700 L3 (during Weeks 1 to 16). The serum haptoglobin also increased during the first seven weeks after the second infection, in three of the four ponies following the second dose of larvae. The serum IgG(T) concentration started to increase from Week 6 or 9 in the ponies given 700 L3, reaching peaks of 44 and 32 g/litre respectively, eight to nine weeks later, compared with a peak of 16 g/litre 20 to 22 weeks after infection in ponies dosed with 200 L3. The IgG(T) concentration increased to a maximum of 25 g/litre in the serum of only one of the four ponies after the reinfection. The serum IgG concentration in all ponies increased nearly twofold during the first eight weeks after both the primary and secondary dose of larvae. It is concluded that the measurement of specific proteins is more reliable and quicker than the electrophoretic separation and quantitation of protein bands, in tracing changes in serum proteins following the artificial infection of ponies with S vulgaris larvae.     

Serodiagnosis of Lyme disease in UK dogs

Lyme disease is a chronic, multisystemic, inflammatory disorder of man and animals associated with infection by the tick-borne spirochaete, Borrelia burgdorferi. Lyme disease was recently reported for the first time in a dog in the UK (May and others 1990). Using an enzyme-linked immunosorbent assay (ELISA), we have performed a serological survey to investigate the prevalence of antibodies to B burgdorferi in UK dogs. The survey has shown that dogs from many areas in the UK have serum antibodies to B burgdorferi, that the presence of serum antibodies is associated with known exposure to ticks and that some dogs seropositive for B burgdorferi have clinical signs consistent with Lyme disease. High levels of serum anti-Borrelia antibodies are not diagnostic for canine Lyme disease, but, in association with appropriate clinical signs, they help to confirm the diagnosis in suspected cases.     

Arthritis and panuveitis as manifestations of Borrelia burgdorferi infection in a Wisconsin pony

Infection with Borrelia burgdorferi caused panuveitis and arthritis in a pony. Spirochetes were detected by direct immunofluorescence in the anterior chamber of the eye. The carpal joints had severe degenerative joint disease, with synovial proliferation. The synovium and serum had B burgdorferi antibody titers of 1:1024. The pony lived in an area of Wisconsin where infection with B burgdorferi is endemic in human beings. Previously, serum antibodies to B burgdorferi had been found in horses, but disease had not been reported.     

Population screening of endangered horse breeds for the foal immunodeficiency syndrome mutation

The Fell and Dales are UK pony breeds that have small populations and may be at risk from in-breeding and inherited diseases. Foal immunodeficiency syndrome (FIS) is a lethal inherited disease caused by the recessive mutation of a single gene, which affects both Fell and Dales ponies and potentially other breeds that have interbred with either of these. FIS, previously known as Fell pony syndrome, is characterised by progressive anaemia and severe B lymphocyte deficiency. The identification of the causal mutation for this disease led to the recent development of a DNA-based carrier test. In this study, the authors used this test to estimate the prevalence of the FIS mutation in the Fell and Dales populations, revealing that approximately 18 per cent of adult Dales ponies and 38 per cent of adult Fell ponies are carriers of the FIS defect. In addition, a study of five potential at-risk breeds was conducted to assess the transfer of the FIS defect into these populations. Of the 192 coloured ponies tested, two were confirmed as FIS carriers: No carriers were found among 210 Clydesdales, 208 Exmoor ponies, 161 Welsh section D, 49 part-bred Welsh section D and 183 Highland ponies.