Corresponding author: Dr Michael R. Lappin, Department of Clinical Sciences, Colorado State University, 300 West Drake Road, Fort Collins, CO 80523; e-mail: firstname.lastname@example.org
Results of Bartonella henselae blood culture, polymerase chain reaction (PCR) assay on blood, or IgG antibody assays do not always correlate with the presence or absence of clinical disease in cats, and B. henselae IgG antibodies in serum do not always correlate with bacteremia. However, little is known concerning Bartonella spp. IgM antibodies in naturally exposed cats.
Bartonella spp. IgM antibodies in serum are associated with fever, stomatitis, and bacteremia based on PCR assay results in experimentally infected or client-owned cats.
Stored sera from cats experimentally infected with B. henselae by exposure to Ctenocephalides felis, client-owned cats with and without fever, and client-owned cats with and without stomatitis were studied.
A Bartonella spp. IgM ELISA was titrated with samples from experimentally infected cats and then test sera from client-owned cats were assayed. Associations among IgM ELISA results, clinical findings, and bacteremia as defined by Bartonella spp. PCR assay were assessed.
All experimentally infected cats developed Bartonella spp. IgM antibodies. Bartonella spp. IgM antibody assay results were not always in agreement with PCR assay results in client-owned cats (60%). Bartonella spp. DNA in blood, IgM antibodies, and IgG antibodies were not associated with the presence of fever or stomatitis.
Conclusions and Clinical Importance
Because Bartonella spp. IgM antibodies as measured by this assay were not associated with fever or stomatitis and were not always in agreement with PCR assay results, there appears to be little need for assessing individual client-owned cats for this antibody class alone.
Bartonella spp. are intracellular bacteria that have been shown to cause disease in people as well as in domestic animals. In people, the most common clinical manifestations of bartonellosis are cat scratch disease, bacillary peliosis, and bacillary angiomatosis. Atypical bartonellosis characterized by a variety of vague and subtle clinical signs such as fever, fatigue, arthralgia, and headache was recently described in a group of immunocompetent people who were occupationally exposed to Bartonella spp.Bartonella henselae, Bartonella clarridgeiae, and Bartonella koehlerae are the most common Bartonella spp. detected in the blood of cats and Ctenocephalides felis is the known or suspected vector for each of the organisms.[4, 5] Although most cats have subclinical Bartonella spp. infections, clinical abnormalities occasionally occur in experimentally infected cats or naturally exposed cats. Fever, uveitis, stomatitis, hematuria, lymphadenopathy, and cardiac abnormalities have been identified most conclusively.[6-15]
Tests for Bartonella spp. previously studied in people and cats include culture, nucleic acid amplification techniques (primarily polymerase chain reaction [PCR] assays), and a variety of serological assays. Although culture and PCR results can be used to document current infection, positive results do not prove that Bartonella spp. was the cause of the clinical syndrome, culture requires up to 6 weeks for results, and PCR assays require special equipment. Detection of IgG antibodies against Bartonella spp. can be used to document exposure but not to prove current infection. Bartonella spp. seroprevalence rates can be very high because of widespread C. felis infestation rates in many countries. For example, in 1 study of cats housed in a shelter in North Carolina, Bartonella spp. antibodies were detected in 93% of the cats tested, most of which were healthy. The high seroprevalence of B. henselae antibodies in healthy cats has contributed to low positive predictive values in most studies of clinically ill cats.[7, 10, 17-19] In addition, Bartonella spp. IgG antibodies do not always correlate with the presence of bacteremia.
Both Bartonella spp. IgG and IgM antibody titers are used to aid in the diagnosis of bartonellosis in humans. Assays to detect specific IgM antibodies also have been used as aids in the diagnosis of other infectious diseases in cats such as toxoplasmosis.[21, 22] Although Bartonella spp. IgM and IgG antibody responses have been described temporally in some experimentally inoculated cats, in those studies infection was initiated by inoculation rather than by exposure to C. felis.[11, 12] In addition, whether Bartonella spp. IgM titers are in agreement with Bartonella spp. bacteremia or are associated with any clinical disease manifestations in naturally exposed, client-owned cats is currently unknown.
The objectives of this study were to optimize a Bartonella spp. IgM enzyme-linked immunosorbent assay (ELISA) for use with feline sera, to apply the optimized assay to sera collected over time from cats experimentally infected with B. henselae from exposure to C. felis, and to determine whether IgM antibodies are associated with bacteremia or common clinical syndromes ascribed to Bartonella spp. infection in cats.
Materials and Methods
Bartonella henselae IgMELISAOptimization
An indirect ELISA was developed to measure the concentration of antibodies against B. henselae lysates. Sera from 4 specific pathogen-free kittens (6 months of age) collected before inoculation (seronegative) and at several time points after IV inoculation of B. henselae (2, 4, 6, and 8 weeks) were selected for use in the initial ELISA titration experiments. The postinoculation samples were assayed in an attempt to identify a sample positive for Bartonella spp. IgM antibodies to use as a positive control sample in the remainder of the experiments. Multiple concentrations of B. henselae lysates, and secondary antibodies as well as various sources and formulations of buffers, blocking solutions and micro ELISA plates were assessed during optimization of the ELISA. During these titrations, it was determined that the largest difference in absorbance values between preinoculation and postinoculation sera was detected during week 4 after B. henselae infection.
In the optimized ELISA, 100 μL of a 1 : 500 dilution of B. henselae lysate in PBS (pH 7.2) was incubated in micro ELISA plates1 for approximately 16 hours at 4°C. Plates were washed 3 times with PBS-Tween solution. One feline serum sample obtained before inoculation and 1 sample obtained at week 4 after IV inoculation with B. henselae were selected for use as positive and negative controls for the remainder of the experiments. A positive control sample, negative control sample, and the sera to be tested were diluted 1 : 64 in PBS-Tween solution, after which 100 μL was pipetted into triplicate wells of a micro ELISA plate; the plate was incubated for 30 minutes at 37°C. Each well then was washed 3 times with 200 μL of PBS-Tween solution. A total of 100 μL of a 1 : 1000 dilution of peroxidase-conjugated goat anti-cat IgM heavy chain specific2 in PBS-Tween solution was pipetted into appropriate wells. After a 30-minute incubation period at 37°C, the plates were washed as described and 100 μL of substrate3 was added to each well. The enzyme reaction was stopped after 10 minutes at approximately 20°C by pipetting 100 μL of 0.18M H2SO4 into each well. The optical density of each well (compared with a substrate control blank) was read at 450 nm with an automated micro ELISA reader.4
The mean absorbance value for the positive control sample, the negative control sample, and each test sample was calculated. The mean absorbance values for all samples were converted to %ELISA units by use of the following formula: (test sample mean absorbance minus negative control sample mean absorbance)/(positive control sample mean absorbance minus negative control sample mean absorbance) × 100 to provide additional standardization among assays as previously reported.
An individual cat was considered positive for B. henselae IgM antibodies if the %ELISA value was greater than the mean IgM%ELISA value plus 3 standard deviations (SD) of the preinoculation samples for a group of 26 specific pathogen free (SPF) adult cats (3 years of age). The Bartonella spp. IgM antibody titer was estimated by comparing the IgM%ELISA value of the sample to a standard curve generated from positive and negative control sera assayed on each plate.
After the initial optimization, the interassay variation of the B. henselae IgM ELISA was estimated by dividing the mean absorbance values of the positive control wells by the mean of absorbance values of the negative control wells for 6 separate IgM ELISA assays and the coefficient of variation of these ratios was calculated (standard deviation/mean × 100).
Experimentally Inoculated Cats
In a separate study, 12 purpose-bred cats had been infected with B. henselae by IV inoculation (n = 6) or exposure to C. felis (n = 6) allowed to infest both groups of cats. In that study, cats infected with B. henselae and SPF cats, respectively, were housed in 2 enclosures separated by mesh. Separation of the cats by mesh walls allowed for fleas to pass between the 2 groups but prevented the cats from having direct contact with one another to lessen the likelihood of B. henselae transmission between enclosures by biting or scratching. Blood and sera then were collected at week 0, week 3, and then weekly for 12 weeks. A Bartonella spp. PCR assay and culture were performed previously on blood and Bartonella spp. IgG ELISA was performed previously on serum.[10, 23] The sera had been stored at −80°C until used in the study described herein. After thawing at room temperature, all sera were assayed in the optimized Bartonella spp. IgM ELISA and the %ELISA calculated.
Naturally Exposed Cats
Sera from healthy cats (n = 51), pair-matched cats with and without stomatitis (n = 108), and pair-matched cats with and without fever (n = 182) had previously been assayed for Bartonella spp. IgG.[10, 19, 24] The Bartonella spp. PCR assay was performed on blood from all the healthy cats and 250 of the 290 cats (86.2%) in the fever and stomatitis groups. The sera had been stored at −80°C until use in the study described herein. After thawing at room temperature, samples with adequate volume (>50 μL) were assayed in the optimized Bartonella spp. IgM ELISA and a titer determined.
Results from the experimentally inoculated cats are reported descriptively. The distribution of the paired sample results from cats with and without stomatitis and cats with and without fever was evaluated using logistic regression, and odds ratios and 95% confidence intervals were determined.5 Wilcoxon's rank sum test was used to determine whether Bartonella spp. IgM ELISA titer magnitude was associated with presence of fever or stomatitis. Significance was defined as P < .05. Percentage agreements among results of the Bartonella spp. IgM ELISA, the Bartonella IgG ELISA, and the Bartonella spp. PCR assay were calculated using data from the naturally exposed cats of the fever study and the stomatitis study when results from all assays were available.[10, 19, 24]
The coefficient of variation of the ratio of positive to negative control absorbance for 6 different ELISAs performed on different days was 4.6%. After all samples in the naturally exposed cats were assayed in the Bartonella IgM ELISA, the data base was searched for all cases with DNA of B. clarridgeiae alone amplified from blood in an attempt to determine whether or not the IgM assay detected antibodies against this Bartonella spp. Of these 12 cats, 9 cats were IgG antibody positive and IgM antibody negative, 1 cat was IgG positive and IgM positive, and 2 cats were negative for both antibodies.
Experimentally Inoculated Cats
All experimentally infected cats developed B. henselae bacteremia as determined both by positive PCR assay results and blood culture. For cats inoculated IV, all cats were Bartonella PCR assay positive on the first test date (week 3). After the initial positive PCR assay result, all IV inoculated cats were PCR positive on subsequent sample dates until week 12 when 2 cats were PCR negative. For cats exposed to fleas, the first positive Bartonella PCR assay result was detected at week 7 (1 cat), week 8 (2 cats), week 9 (1 cat), week 10 (1 cat), or week 11 (1 cat). After the initial positive PCR assay result, all cats exposed to fleas were PCR positive for the duration of the study. Group mean IgG%ELISA and IgM%ELISA results over time are shown in Figure 1.
None of the IV inoculated cats developed clinical illness over the course of the study. Of the 6 cats exposed to B. henselae from C. felis, 3 developed fever and inappetence in week 10 of the study, necessitating antibiotic therapy as dictated by the study design. One of the 3 cats with fever was euthanized in week 10 of the study because of the severity of clinical illness and was shown to have Bartonella-associated myocarditis on necropsy and histopathology. All 6 cats were PCR positive for B. henselae DNA in blood at the time fever was detected in the 3 cats. Although there were no statistical differences in Bartonella IgM or IgG concentrations between the cats with and without fever in the experimentally inoculated group, the cat that died had the lowest IgM%ELISA (35.4% versus a range of 45.3–130.6% in the other 5 cats) and was the only cat that did not develop detectable Bartonella spp. IgG concentrations.
Naturally Exposed Cats
The prevalence rates for Bartonella spp. IgM, IgG, and DNA in the healthy shelter cats were 13.7, 86.3, and 56.9%, respectively.
There were no significant differences in Bartonella spp. IgM, IgG, or DNA prevalence rates between the pair-matched samples from cats with and without fever and with and without stomatitis (Table 1). Similarly, Bartonella spp. IgM titer magnitude was not associated with fever or stomatitis (Table 2). In this analysis, the median titers frequently were 0, and consequently mean results are presented. The magnitude of Bartonella spp. IgG titers was significantly higher in healthy controls than in cats with fever (P = .0241).
Table 1. Associations between Bartonella spp. test results and the presence of fever or stomatitis in naturally exposed cats.
Results determined in pairs by logistic regression. In the analysis design, OR values greater than 0 were detected when affected cats were negative and control cats were positive, and OR values less than 0 were detected when affected cats were positive and control cats were negative. P < .05.
Table 2. Associations between Bartonella spp. IgM and IgG titer magnitude and fever or stomatitis in naturally exposed cats.
Wilcoxon's rank sum test with significance defined as P < .05.
Bartonella spp. Assay Agreements
The IgM, IgG, and PCR assay results were available for 250 of the cats in the fever and stomatitis studies. When the Bartonella spp. IgM and PCR assay results were compared, 60.0% (150 cats) had agreement between ELISA and PCR assay results; 9 of 250 cats (3.6%) were both IgM and PCR positive and 141 of 250 cats (56.4%) were both IgM and PCR negative. Of the 250 cats, 81 (32.4%) were IgM positive but PCR negative and 19 (7.6%) were IgM negative but PCR positive. When the Bartonella spp. IgG and PCR assay results were compared, 59.6% (149 cats) had agreement between ELISA and PCR assay results; 21 of 250 cats (8.4%) were both IgG and PCR positive and 128 of 250 cats (51.2%) were both IgG and PCR negative. Of the 250 cats, 94 (37.6%) were IgG positive but PCR negative and 7 (2.8%) were IgG negative but PCR positive.
Potential benefits of utilizing Bartonella spp. serological assay results include the ability to receive results in a timely manner when compared with culture as well as avoiding potential quality control issues that can be of substantial concern with PCR analysis. Because Bartonella spp. IgG assay results frequently are not associated with clinical disease syndromes or bacteremia, we chose to evaluate Bartonella spp. IgM in serum in the study described herein.
There was minimal interassay variation in the IgM ELISA assay. The secondary antibody was heavy chain specific and thus the assay should only detect IgM antibodies against Bartonella spp. Both B. henselae and B. clarridgeiae were detected by IgG antibodies in the previous study of the cats with and without fever. In the study described herein, only 1 of 12 B. clarridgeiae PCR positive cats was Bartonella spp. IgM positive but 9 of 12 cats were positive for Bartonella IgG. Thus, although B. henselae was used as the antigen source, the IgM assay also may detect antibodies against B. clarridgeiae. In at least 1 other study, possible cross reactivity between these 2 species also was shown. Alternately, the B. clarridgeiae PCR positive cats that were positive in the B. henselae-based assay may have been coinfected by B. henselae below the detectable limits of the PCR assay or had been previously infected by B. henselae.
The temporal appearance of Bartonella spp. IgM and IgG in the serum of the IV inoculated cats described herein was similar to previous studies in which cats were inoculated IV or intradermally.[12, 13, 26] In each of those studies, IgM antibodies in parenterally inoculated cats were generally detected before IgG, were positive frequently within 1 week of inoculation, and then declined quickly over several weeks. Peak IgG antibody responses took several weeks to develop in some cats and antibodies were maintained at high concentrations for many weeks before a gradual decline, sometimes up to few months later.[12, 13, 24] In the instances in which the serum antibody responses were not robust or were delayed, the authors speculated strain or dose-related effects may have occurred.[12, 13]
In the cats exposed to C. felis in this study, the initial detection of B. henselae DNA in blood did not occur until 7 weeks after flea exposure, with IgM and IgG antibodies being detected shortly thereafter. This delay in developing bacteremia and subsequent seroconversion is likely a dose effect with flea exposure likely providing a lower dose of B. henselae than studies that used IV or intradermal inoculation of cultured organisms. In contrast to cats inoculated IV or intradermally, IgM serological responses in the cats exposed to C. felis sometimes lagged behind IgG responses and IgM antibodies were detectable for at least 4 weeks after initial detection. Because IgM antibodies were detected in C. felis exposed cats until the study endpoint, the maximal duration of this response is unknown. The cause of the apparent IgM persistence is unknown, but may relate to the route of inoculation, dose, or the strain of B. henselae as previously noted.
Although the B. henselae IV inoculated cats described herein were clinically normal throughout the study, 3 of the 6 cats that were infected by exposure to C. felis developed clinical signs of illness with 1 cat requiring euthanasia because of disease severity. Whether or not this relates to pathogenic effects associated with vector transmission should be explored in future studies. There were no obvious differences in IgG, IgM, and PCR results between the cats with and without illness in the C. felis-exposed group, but the sample sizes were small and consequently statistical comparisons were not made. The cat that required euthanasia was the only animal that did not develop an IgG response and had the lowest IgM response of all cats. Although this cat was negative for FeLV antigen and FIV antibodies, it is possible it may have had underlying immunodeficiency or genetic predisposition that could have complicated the Bartonella infection. This outcome may have related in some way to failure of the humoral immune response that then precipitated serious clinical disease. People with concurrent diseases, most notably diseases associated with immunocompromise such as AIDS, are more likely to develop serious complications with Bartonella infection. These individuals may be more prone to developing systemic manifestations of disease and long-term complications, such as bacillary angiomatosis because of impaired cell-mediated immunity. The cat described herein may have been more bacteremic than others that failed to develop clinical illness.
In studies of Toxoplasma gondii infections of cats, concurrent evaluation of IgM and IgG responses has been used to document acute infection, and detection of IgM antibodies correlates with clinical illness in some cats.[21, 22] Fever and stomatitis are 2 syndromes frequently suspected to result from Bartonella spp. infection in cats, and thus we chose these syndromes to evaluate for associations with Bartonella spp. IgM test results.[10, 17] Similar to previous studies with Bartonella spp. IgG, detection of Bartonella spp. IgM or magnitude of IgM response did not correlate with either clinical syndrome (Tables 1 and 2). Many of the control cats were positive for Bartonella spp. IgM antibodies. Because these cats were only sampled once, the duration of IgM positive test results or Bartonella spp. infection was unknown. These cats may have been recently infected. Failure of Bartonella IgM antibodies to correlate with clinical disease may relate to the persistence of IgM in otherwise normal cats exposed to fleas. It is also possible that these syndromes are not caused by Bartonella spp. infections. Based on these results, there appears to be no indication to add Bartonella spp. IgM assay to the current diagnostic evaluation of cats with suspected bartonellosis.
Bartonella spp. bacteremia can be confirmed by culture and suggested by amplification of Bartonella spp. DNA from blood. However, culture can require weeks for results to return, thus serum antibody responses have been used in an attempt to predict bacteremia by comparing antibody response with culture or PCR results in previous studies.[1, 10, 19, 24] In this study, the IgM ELISA and PCR results were in agreement for only 60% of the 250 samples from cats with or without fever or stomatitis. In these cats, 32.4% of the IgM positive cats were PCR negative. In addition, 7.6% of the IgM seronegative cats were PCR positive. Results were similar for the IgG ELISA results. Thus, serological test results frequently did not agree with the presence or absence of bacteremia as has been described in many other studies and support the recommendation that serological test results are not be used to determine the Bartonella spp. infection status of individual cats.[1, 10, 19, 24]
Funding provided by the Center for Companion Animal Studies at Colorado State University. The authors thank Dr Steven Radecki for the statistical analyses used in this study.
Immulon 2, Thermo Fisher Scientific, Waltham, MA
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