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.
- Top of page
- Materials and Methods
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]