Bartonella species are zoonotic agents and primary pathogens in cats. Hyperglobulinemia has been associated with bartonellosis in humans and cats.
Bartonella species are zoonotic agents and primary pathogens in cats. Hyperglobulinemia has been associated with bartonellosis in humans and cats.
To evaluate for associations between Bartonella species immunoglobulin G (IgG) antibodies and serum biochemistry panel results in privately owned cats.
1,477 privately owned cats.
Residual sera were collected after biochemical evaluation for this prospective, cross-sectional serosurvey. Bartonella species IgG ELISA was performed with a cutoff value of ≥1 : 64. Stepwise logistic regression analysis was performed with the endpoint titer as the outcome variable. The final statistical model included age, albumin, ALP activity, ALT activity, bilirubin, creatinine, glucose, and globulin as covariates. Serum protein electrophoresis was performed with serum from 50 cats with and without antibodies to Bartonella species and hyperglobulinemia. Sera from cats seropositive to Bartonella species and with hyperglobulinemia were assessed for evidence of exposure to other infectious agents associated with hyperglobulinemia.
Risk of seropositivity to Bartonella species was positively associated with the natural log of globulin concentration (OR = 11.90, 95% CI 6.15–23.02, P < .0001), and inversely associated with the natural log of glucose concentration (OR = 0.66, 95% CI 0.50–0.87, P = .004). Another explanation for hyperglobulinemia was not identified for most cats with Bartonella species antibodies. Hyperglobulinemia was primarily caused by polyclonal gammopathy in cats that were seronegative and seropositive for Bartonella species.
Hyperglobulinemia was significantly associated with seropositivity to Bartonella species. Testing for bartonellosis is warranted in cats with unexplained hyperglobulinemia and clinical or laboratory findings suggestive of bartonellosis.
Interest in Bartonella species (spp.) has been increasing as more is discovered about their role as primary pathogens and zoonotic agents.[1, 2]Bartonella spp. are recognized as primary pathogens in human diseases, including cat scratch disease, bacillary angiomatosis, endocarditis, and bacillary peliosis.[1-3]Bartonella infection has been associated with extended, often asymptomatic, intraerythrocytic bacteremia in domestic cats,[4-8] the primary mammalian reservoir host for Bartonella henselae. In addition, Bartonella spp. DNA has been amplified from the oral cavities and nail beds from 20 to 51% of feral and pet cats.[9-11] Increased exposure to cats, particularly kittens, and cat-related trauma have been associated with a higher prevalence of Bartonella-associated disease in people.[2, 3, 12]
Elucidation of potential associations between Bartonella infection and clinical disease in cats is complicated by variability in host susceptibility, virulence of different isolates, and disease severity. Many cats exposed to or infected with Bartonella spp. do not develop clinical disease, but some cats develop fatal sequelae from infection.[1, 3] Lethargy, anorexia, fever, endocarditis, myocarditis, lymphadenopathy, neurologic disorders, hematuria, and uveitis have been associated with experimental and naturally occurring bartonellosis in cats.[9, 13-18] Conflicting data exist for a potential role for Bartonella spp. bacteremia for gingivostomatitis and hepatic disease in cats.[19-24]
Experimentally infected cats have been shown to have infiltrates of lymphocytes and plasma cells in a number of tissues, including the feline liver, pancreas, and kidneys.[15, 18] Because serum biochemical panel results were not reported in those studies, it remains unknown whether these inflammatory infiltrates were associated with laboratory evidence of disease. To our knowledge, only 1 study has evaluated for correlations between Bartonella spp. serology results and laboratory abnormalities in client-owned cats. In that study, the only associations identified were between antibodies to Bartonella spp. and hematuria and lymphocytosis. Hyperglobulinemia, including monoclonal and biclonal gammopathies, has been documented in some people with bartonellosis,[26, 27] in a cat with osteomyelitis caused by B. vinsonii, and in a cat with B. henselae associated endocarditis.
The primary objective of this study was to evaluate for associations between the presence of antibodies to Bartonella spp. and serum biochemical results, including globulin concentrations, using serum from client-owned cats. The secondary objective was to characterize hyperglobulinemia in cats with seropositivity to Bartonella spp. and evaluate for potential concurrent infection with agents that induce chronic infections and, thus might contribute to development of hyperglobulinemia.
Residual serum samples were obtained from a commercial diagnostic laboratory1 for 1,477 cats on which a biochemical profile had been performed. Albumin, bilirubin, BUN, calcium, chloride, cholesterol, creatinine, globulin, glucose, phosphate, potassium, sodium, and total protein results, as well as activities of serum alanine aminotransferase (ALT), alkaline phosphatase (ALP), aspartate aminotransferase (AST), and creatine kinase (CK) were available for all cases. Age was available for 1,348 cases. Because samples were obtained from a commercial diagnostic laboratory in lieu of sample disposal, signalment, vaccination history, and clinical findings were not available. Geographic distribution was also not available for individual cases, but the majority of samples tested in this laboratory originated from the northeastern United States. Samples were stored at −70°C until utilized in this experiment.
Samples were assayed by an ELISA for B. henselae immunoglobulin G (IgG) antibodies as previously described. Results reflect Bartonella spp. exposure, not B. henselae specifically, because of cross-reactivity with other Bartonella spp. Plates were read on an automated microELISA plate reader at 450 nm. The optical density of all sample and control wells was compared with the substrate control blank. Samples were run in triplicate wells, whereas controls were run in quadruplicate wells. A standard curve was generated with the positive and negative optical density results to determine an endpoint titer for suspect samples. A positive cutoff value of ≥1 : 64 was used in this study.
To further characterize the association between antibody titers to Bartonella spp. and hyperglobulinemia (see 'Results'Results below), protein electrophoresis was performed with serum from 50 cats: 20 cats with hyperglobulinemia and antibody titers ≥1 : 128 to Bartonella spp. (Group 1), 20 cats with hyperglobulinemia and negative antibody titers to Bartonella spp. (Group 2), and 10 cats with normal globulin levels and negative antibody titers to Bartonella spp. (Group 3). The ≥1 : 128 cutoff for antibody titers to Bartonella spp. used for selection of Group 1 was chosen arbitrarily in an attempt to exclude any cat that might have been falsely positive at the threshold cutoff value of the assay (1 : 64). Serum protein electrophoresis was performed by a commercial kit as per manufacturer's instructions.2,3 Albumin, alpha 1, alpha 2, beta, and gamma globulin concentrations were recorded for each cat. Serum protein electrophoresis gels were reviewed by 2 investigators (JDS and JCW) to characterize elevations.
The samples from Group 1 were further evaluated for other agents associated with chronic infections in cats and, thus potentially associated with development of hyperglobulinemia. Each serum sample was centrifuged at 10,000 × g for 15 minutes, and the serum pipetted from the pellet. Serum was then assayed for feline leukemia virus antigen,4 feline immunodeficiency virus antibody,4 and Dirofilaria immitis antigen,4 as well as for IgM and IgG antibodies against Toxoplasma gondii.3 The pellet was resuspended in PCR buffer from a commercially available kit and the total DNA extracted as per the manufacturer's instructions.5 The total DNA was assessed by previously reported PCR assays for DNA of GAPDH (DNA control), Bartonella spp., Mycoplasma haemofelis and haemominutum, Ehrlichia spp., Anaplasma spp., and Wolbachia spp.3,30–32
Descriptive statistics were generated for each biochemical parameter, and samples were analyzed for normality by the Kolmogorov-Smirnov test. Non-normally distributed data were logarithmically transformed, before statistical analyses. Stepwise logistic regression analysis was performed with serologic status (seropositive versus seronegative), as the outcome variable. Collinearity of the explanatory variables was evaluated, before the evaluation of the association of antibodies to Bartonella spp. and biochemical analytes. Assuming no collinearity of explanatory variables, the statistical model initially included age, albumin, ALP activity, ALT activity, bilirubin, SUN, creatinine, globulin, and glucose as covariates. For all analyses, P < .05 was considered significant. Odds ratios (OR) and 95% confidence intervals (CI) were calculated for significant variables. Data were analyzed by use of a commercially available software package.6
A total of 590 (40%) of 1,477 samples were seropositive for antibodies to Bartonella spp. Concentrations for glucose, creatinine, bilirubin, and globulin, as well as ALP and ALT activities, were log transformed before statistical analysis owing to non-normal distribution. SUN was excluded from the statistical model owing to collinearity with creatinine. Age, albumin, ALT activity, ALP activity, bilirubin, and creatinine were excluded by the stepwise logistic regression as unrelated to Bartonella spp. ELISA results. Presence of antibodies to Bartonella spp. was negatively correlated with the natural log of glucose concentration (P = .004; OR 0.66, 95% CI 0.50–0.87), and positively correlated with the natural log of globulin concentration (P < .001; OR 11.90, 95% CI 6.15–23.02). Based on back transformation of the data, a decrease in glucose concentration by 1 mg/dL, was associated with a 0.243-fold increase in the risk of seropositivity to Bartonella spp. Similarly, an increase in globulin concentration by 1 mg/dL was associated with a 4.37-fold increase in the risk of seropositivity to Bartonella spp. Globulin concentrations were above the upper limit of the reference interval (5.6 mg/dL) for 66 samples (4.46%). The prevalence of hyperglobulinemia was 2.8% (25/887) in seronegative cats and 6.9% (41/590) in seropositive cats.
Albumin, alpha 2, beta, and gamma globulin concentrations were normally distributed for all groups. Alpha 1 globulin concentrations were non-normally distributed in Group 1 but were normally distributed for Groups 2 and 3 (Table 1). Hyperglobulinemia was caused by polyclonal gammopathy in 19/20 cats (95%) in Group 1, and 18/20 cats (90%) in Group 2. One cat in Group 1 had a sharp beta-globulin peak; 2 cats in Group 2 had monoclonal gamma globulin peaks.
|Analyte||Group 1||Group 2||Group 3|
|Alpha 1 globulins (g/dL)||0.50||0.36–1.53||0.54||0.16–1.58||0.50||0.34–0.64|
|Alpha 2 globulins (g/dL)||1.31||0.61–4.62||1.35||0.71–2.69||1.64||1.04–2.35|
|Beta globulins (g/dL)||0.88||0.51–4.54||0.95||0.62–2.66||0.73||0.41–1.66|
|Gamma globulins (g/dL)||3.61||0.61–5.98||4.43||2.81–6.69||1.66||0.24–2.55|
GAPDH was amplified from the pellet from 8 of 20 samples with seropositivity to Bartonella spp. and hyperglobulinemia. All samples were negative for DNA of Bartonella spp., Mycoplasma spp., Ehrlichia spp., Anaplasma spp., and Wolbachia spp. FIV antibodies were detected in 3 samples, and t. gondii IgG antibodies were detected in 4 samples (titers = 1 : 64, 1 : 64, 1 : 128, and 1 : 256). One sample was positive for Bartonella spp. IgG, Toxoplasma IgG, and FIV antibodies concurrently.
The prevalence of antibodies to Bartonella spp. in this study (40%) was consistent with results from previous reports.[1, 9, 14, 19, 24, 25, 30, 33-38] Results of this study demonstrated a strong positive correlation between seropositivity with Bartonella spp. and hyperglobulinemia in this population of privately owned cats. For every 1 mg/dL increase in globulin concentration, the likelihood of having a positive Bartonella spp. antibody titer increased 4.37-fold. Based on subgroup analysis, hyperglobulinemia was primarily because of polyclonal gammopathy. A mild inverse correlation of unknown clinical relevance was present between the natural log of glucose concentration and seroreactivity to Bartonella spp. No other significant associations were identified between biochemical analytes and seropositivity to Bartonella spp.
The strong association between positive antibody titers to Bartonella spp. and hyperglobulinemia in this study is not surprising given the prolonged bacteremia that can be associated with infection by Bartonella spp. Studies of experimental Bartonella spp. infection in rats have demonstrated that inoculated bacteria are rapidly cleared from the bloodstream followed by a recrudescent bacteremia on day 4 or 5. After re-entering the bloodstream, organisms adhere to and invade circulating erythrocytes. Bartonella spp. also show a tropism for endothelial cells, replicating in them after adherence and engulfment.[40, 41] Based on results of experimental studies, Bartonella spp. infection triggers a proinflammatory cascade mediated by NF-κΒ that results in neutrophils rolling on and adhering to infected endothelial cells.Bartonella spp. lipopolysaccharide binds to Toll-like receptor 2 on monocytes and dendritic cells, stimulating the production of cytokines and chemokines including tumor necrosis factor α and interleukin 10. In humans, the subsequent humoral response involves induction of IgG1 and IgA antibodies. The transition from acute to chronic inflammation is postulated to involve recruitment of macrophages in response to the release of monocyte chemoattractant protein-1 by activated endothelial cells. As lymphocytosis has been associated with B. henselae antibodies in cats, it is possible that similar mechanisms could result in hyperglobulinemia in cats.
While a small number of naturally exposed humans and cats with bartonellosis have hyperglobulinemia,[26-29] this association has not been made in studies of experimentally infected cats.[5, 6, 13, 15, 17, 18] Because cats are a host-adapted reservoir for Bartonella henselae, koehlerae, and clarridgeiae, infected cats might not develop hyperglobulinemia from these infections alone. However, it is possible that infection with more than 1 Bartonella spp., with more pathogenic Bartonella spp. or strains, or with other coinfections could play a role. Wide genomic variability within Bartonella isolates has been documented, reflecting in vivo adaptation to host pressures, including humoral immunity.Bartonella coinfections and serial reinfection have been documented in cats, both of which could contribute to development of polyclonal gammopathy.[7, 45] In this study, only serum samples were available for assay. After repeat centrifugation of the serum in an attempt to collect cell associated DNA in the pellet, we were only able to amplify GAPDH as a positive DNA control from 8 samples. Bartonella spp. DNA was not amplified from these 8 samples. Thus, we cannot accurately assess the role that coinfection with multiple Bartonella spp. might have played in these samples. In this study, we also evaluated for evidence of other coinfections in a subset of Bartonella spp. seropositive cats. Antibodies against FIV and T. gondii were detected in several cats, as has been previously reported, which might have played a role in development of hyperglobulinemia.[21, 22, 33, 38]
In this study, all but one of the Bartonella spp. seropositive cats studied had polyclonal gammopathies. The remaining seropositive cat had a monoclonal beta-globulin gammopathy, similar to that reported in a dog with bartonellosis caused by B. henselae infection. Monoclonal and biclonal IgG kappa gammopathy has also been detected in humans with bartonellosis.[26, 27] Unfortunately, because only a small number of cats were assessed by serum protein electrophoresis in this study and clinical diseases present in the cats are not known, the potential for Bartonella spp. infection to cause monoclonal gammopathies in the cat remains undetermined. The cause of the monoclonal gamma globulin peaks in the 2 seronegative cats with hyperglobulinemia is also unknown.
Because of its indolent nature and ability to cause monoclonal gammopathy in humans, it has been speculated that chronic antigenic stimulation and clonal expansion secondary to bartonellosis might have a role in the development of plasma cell tumor or lymphoma in humans. In 1 cat with recurrent B. vinsonii osteomyelitis, bony infiltration with plasma cells was profound enough to warrant consideration of plasma cell tumor on 2 separate biopsies performed 15 months apart.Bartonella spp. infection is common in golden retrievers with lymphoma. These findings combined with the association of B. henselae antibodies with lymphocytosis in naturally exposed cats suggests that future studies for potential associations between Bartonella spp. infections and lymphoma or multiple myeloma in cats should be undertaken.
In this study, an association between antibodies to Bartonella spp. and age was not made. Conflicting data exist regarding associations between age and Bartonella spp. antibody prevalence rates in cats.[5, 19, 35-37] The majority of the studies reported to date have had small sample numbers or have varied widely in age distribution, which could have affected the results. Based on careful evaluation of prevalence data by age for the other reported studies, it appears that seroprevalence increases with age until cats reach 1–3 years of age, after which it plateaus or declines. The use of serial Bartonella spp. PCR assays and cultures in future studies would aid in clarifying the impact of bacteremia and host-adaptation on age-associated changes in seropositivity.
There were a number of limitations to this study. Because serum samples were collected in lieu of disposal, it remains unknown how many of the cats were indoor only had had exposure to fleas were receiving flea prophylaxis or had clinical signs of disease. Because serological cross-reactivity exists between B. henselae and other Bartonella spp., results do not document a specific correlation between hyperglobulinemia and B. henselae antibody development. In addition, causality cannot be determined based on analysis of cross-sectional data. These results do not prove that hyperglobulinemia occurs secondary to bartonellosis or specifically reflects Bartonella spp. antibody concentrations, merely that cats with hyperglobulinemia are more likely to be seropositive for antibodies to Bartonella spp. Because not all cats with Bartonella bacteremia develop detectable antibodies to Bartonella spp., it is also possible that some cats in Group 2 had underlying bartonellosis. Finally, Bartonella spp. isolation by culture was associated with gingivostomatitis in 1 study of privately owned cats, whereas a similar association was not made with antibodies to Bartonella spp., underscoring the limitations of relying on 1 diagnostic assay to elucidate potential associations between Bartonella spp. and clinical disease. Prospective evaluation of Bartonella spp. serology in concert with PCR assays, culture, and species and strain identification in a large population of privately owned cats with known housing status, flea exposure, clinical signs, and diagnostic findings is warranted to determine the clinical relevance of these findings.
Given the wide range of diseases for which Bartonella spp. are postulated to have an etiopathologic role, it can be challenging to know when evaluation of Bartonella serology is warranted. In addition, interpretation of titers is complicated by the high prevalence of Bartonella seroreactivity and bacteremia in clinically normal cats. For example, differences in Bartonella spp. antibody prevalence rates were not identified in recent case-control studies of gingivostomatitis or fever.[19, 23, 24, 30] In addition, Bartonella spp. seropositive rates were higher in healthy control group cats than in cats with uveitis or neurologic disease.[36, 37] However, based on the results of this study, testing for bartonellosis as described above may be warranted in cats with unexplained hyperglobulinemia, particularly if clinical findings previously associated with bartonellosis and exposure to C. felis are present.
The work was funded by the Center for Companion Animal Studies, Department of Clinical Sciences, Fort Collins, CO. The authors thank Antech Diagnostics, Lake Success, NY for providing the sera and IDEXX Laboratories in Portland, ME for donating some of the assays used in the study.
Antech Diagnostics, Lake Success, NY
Hydragel 7-Protein and Sebia Hydrasys, Sebia Inc, Norcross, GA
Veterinary Diagnostic Laboratory, Colorado State University, Fort Collins, CO
SNAP Triple, IDEXX Laboratories, Portland, ME
QIAamp DNA Blood Mini Kit, Qiagen Inc, Valencia, CA
Statview for Windows v.5.0.1, SAS Institute, Inc, Cary, NC