Experimental infection and horizontal transmission of Bartonella henselae in domestic cats

Infecção experimental e transmissão horizontal de Bartonella henselae em gatos domésticos

EXPERIMENTAL INFECTION AND HORIZONTAL TRANSMISSION OF Bartonella henselae IN DOMESTIC CATS

Marcelo de Souza ZANUTTO(¹), Elza Masae MAMIZUKA(²), Roberto RAIZ-JÚNIOR(³), Thais Martins de LIMA(²), Constância Lima DIOGO(³), Thelma Suely OKAY(³) & Mitika Kuribayashi HAGIWARA(¹)

SUMMARY

In order to study B. henselae transmission among cats, five young cats were kept in confinement for two years, one of them being inoculated by SC route with B. henselae (10⁵ UFC). Only occasional contact among cats occurred but the presence of fleas was observed in all animals throughout the period. Blood culture for isolation of bacteria, PCR-HSP and FTSZ (gender specific), and BH-PCR (species-specific), as well as indirect immunofluorescence method for anti-B. henselae antibodies were performed to confirm the infection of the inoculated cat as well as the other naive cats. Considering the inoculated animal, B. henselae was first isolated by blood culture two months after inoculation, bacteremia last for four months, the specific antibodies being detected by IFI during the entire period. All contacting animals presented with bacteremia 6 months after experimental inoculation but IFI did not detect seroconversion in these animals. All the isolates from these cats were characterized as Bartonella (HSP and FTSZ-PCR), henselae (BH-PCR). However, DNA of B. henselae could not be amplified directly from peripheral blood by the PCR protocols used. Isolation of bacteria by blood culture was the most efficient method to diagnose infection compared to PCR or IFI. The role of fleas in the epidemiology of B. henselae infection in cats is discussed.

KEYWORDS: Cat; Felines; Bartonella henselae; Bartonella quintana; Hemoculture; Polymerase Chain Reaction; Indirect immunofluorescence.

INTRODUCTION

Cat-scratch disease (CSD) is transmitted to human beings by scratching, licking or bitting of cats harboring B. henselae. Also, fleas (Ctenocephalides felis) are thought to be involved in transmission. After infection of fleas, fecal elimination of viable bacteria occurs¹¹. Bacteremia has already been reported in catsafter intradermal inoculation of feces from contaminated fleas⁸.

Classic CSD is characterized by regional lymphoadenopathy, which corresponds to the draining route of bacteria inoculation site. Atypical CSD may occur, mainly in immunodepressed patients, as bacillary angiomatosis and peliosis, endocarditis, recurrent fever and bacteremia with ocular, hepato-splenic, skeletal, cutaneous or pleuro-pulmonar symptoms⁶.

The cat is the reservoir, and fleas are possibly the vectors of bacteria. The cat is the only animal undoubtedly involved in Bartonella henselaetransmission^1,5. Young animals less than one year old, are the animals presenting the highest transmission potential and are generally asymptomatic^1,20. Occasional fever, lethargy, regional lymphoadenopathy, edema and abscess formation at the site of inoculation^1,17,18,20, besides histological alterations characterized by lymphoplasmocytic infiltrate in liver, kidneys, lymphonodes and heart occur in chronically affected animals¹³.

Infected felines bacteremia may last for a variable period of time, and can also be intermittent^{1,5,8,13,14,17,18,20}. Isolation of Bartonella is done by blood culture even though bacteria growth in appropriate medium is fastidious. After natural or experimental infection, cats may develop humoral immunity with specific IgG antibodies production detected by enzyme-linked immunosorbent assays or IFI^1,5. Serological tests specificity seems to be unsatisfactory due to cross reactions with B. quintana antigens (etiological agent of trench fever, bacillary angiomatosis and peliosis, transmitted to man by lice)^15,19. Frequently, antibody anti-B. henselae and anti-B. quintana, can both be found in the same animal, pointing out the low specificity of IFI^16,20.

The identification of species and strains of Bartonella sp is based on molecular characterization of isolates or in infected clinical samples^{2,5,8,9,12,13,14,17,18,20}. Therefore, PCR, a more sensitive method in comparison to hemoculture, might be used to detect Bartonella and type species¹³.

Because of the unique role of cats in the epidemiology of CSD disease, the aim of this study was to analyze the susceptibility of young cats to Bartonella henselae infection, as well as its horizontal transmission, so that blood cultures were used to isolate B. henselae, three different PCR protocols were used to detect and characterize isolates, and IFI detected serum specific anti-B. henselae antibodies thus confirming seroconversion.

MATERIAL AND METHODS

Animals: Five healthy, naive cats were kept, from 2 months old until 2 years, in a confined environment, with sporadic contact among them. All animals were free of parasites, immunized against Panleukopenia, Herpesvirus, Calicivirus and Rabies. At the age of 6 months, one cat was challenged with 10⁵CFU of viable B. henselae (Houston strain), by SC route and submitted to clinical evaluation every 2 – 3 days, for one year. Blood samples were collected asseptically by jugular venipuncture for hemoculture (1.0 mL in Isolator pediatric tubes Oxoid^®)³, PCR (1.0 mL in EDTA tubes) and IFI (1.0 mL in gel separator tubes) on days 7, 14, 30, 54, 68, 90, 120, 150, 185, 210, 240 and 360 after inoculation. The remaining cats were submitted to physical examination and blood sampling at age 2, 4, 6, 12, 18, 20, 22 and 24 months.

Hemoculture: Blood (1.0 mL) was transferred from isolator pediatric tube (Oxoid^®) into microtubes and centrifuged at 503 g for 3 minutes; 50 µL of sediment were placed on brain-heart infusion agar (Oxoid^®) supplemented with 5% of defibrinated sheep blood³. Plates were kept at 35 ^oC in a 5% CO₂ atmosphere (CO₂ Incubator Sanyo^® - model MCO-15 A). The presence of bacterial growth was assessed weekly during five weeks.

Polymerase Chain Reaction: DNA extraction was performed in blood samples according to the technique described by FERRIE et al.⁷. A scrapping of all colonies was used to extract DNA from the isolate. Briefly, 500 µL of a NaOH solution (50 mM) were added to the sample which was then incubated at 100 ^oC for 5 minutes; centrifuged at 15,300 g at 4 ^oC for 4 minutes; and 100 µL of TRIS-HCl (1M, pH 7.5) were added. The final concentration of DNA in the supernatant used was 1 µg/50 µL and 10 ng/50 µL for peripheral blood and isolates respectively.

HSP and FTSZ-PCR protocols, which amplify a segment of the gene that encodes a 60KDa heat shock protein (HSP), producing a 414bp segment², and a gene segment that encodes the cell division protein FtsZ, producing a 1,580 bp product, respectively¹², as well as, a pair of species-specific primers (BH-PCR) which amplifies a 354 bp fragment of B. henselae (also belongs to the FtsZ gene¹²), were used.

To a 50 µL of final volume, 200 µM dNTP, 0.4 µM of each primer, 2.5U of Taq DNA polymerase (Life Technologies^®), 1X (200 mM Tris-HCl, 500 mM KCl, pH = 8.4) buffer, 1.5 mM of MgCl₂ (FTSZ and BH-PCR) and 2 mM (HSP-PCR) were added. Amplifications were performed in a PTC-150 Minicycler (MJ Research"): HSP-PCR began with a initial denaturation step (94 ^oC for 5 minutes), followed by 35 cycles of 94 ^oC for 1 minute, 55 ^oC for 2 minutes and 72 ^oC for 3 minutes, and a final extension at 72 ^oC for 10 minutes. FTSZ and BH amplifications were performed as follows: 30 cycles of 94 ^oC for 1 minute, 60 ^oC for 1 minute (FTSZ-PCR) and 55 ^oC for 1 minute (BH-PCR), and 72 ^oC for 3 minutes, followed by a final extension at 72 ^oC for 30 minutes.

Primers NS-31 and NS-41 that amplify a 652 bp fragment of a gene encoding a 18S ribosomal subunit of all eukaryotic organisms, were used to ensure the quality of extracted DNA¹⁰.

As the positive control, DNA extracted from B. henselae Houston strain and B. quintana Oklahoma strain were used. Negative controls consisted of sterile distilled water instead of DNA.

HSP amplified products were submitted to a 2% agarose gel electrophoresis, and 1% agarose gels for FTSZ and BH (Life Technologies^®), in a TAE X1 buffer, for 1 hour at 80V. Molecular weight standards of 100 bp and 1Kb were used (Life Technologies^®). Gels were stained with ethidium bromide (0.5 µg/mL), visualized in a UV transiluminator (Hoefer - Amersham Pharmacia Biotech^®), and photographed using a Polaroid Camera (Gel Cam Sigma^®), T-667 films (Polaroid^®).

Indirect Immunofluorescence Test (IFI): IFI was done according to CAMARGO⁴. Bartonella henselae Houston strain (co-cultivated in VERO cells, National Reference Center in Rickettsiosis, Marseille, France), was used as antigen, and sheep globulin anti-feline IgG (Sigma^®), marked by fluoresceine isothiocyanate as the conjugate. Anti-B. henselae hyperimmune rabbit serum and the respective anti-rabbit IgG control (Sigma^®) were used as positive controls. The antibody titer of a sample was considered to be the highest dilution in which fluorescence could be observed. Antibody titer ³ 64 was adopted as the cut-off value for IFI reaction because sensitivity and specificity of the technique are adequate when titers are ³ 64¹⁹.

RESULTS

B. henselae inoculation by SC route in one of the cats (animal 1) resulted in bacteremia and the isolation of the agent by hemoculture two months (68 days) after inoculation. Morphology and staining characteristics of the colonies (Fig. 1), were suggestive of Bartonella sp, and identity of the isolate was confirmed by PCR, using gender specific primers (2 protocols) (Fig. 2) and species-specific primers (1 protocol). Bacteremia lasted for 120 days, as seen in Table 1. Serum specific antibodies (IFI) were detected 30 days after inoculation, with IgG titers rising and falling in parallel with bacteremia (Table 1).

Bacteremia was observed in all cats that shared the same environment beginning four months after the inoculation of the first animal and present for a variable period of time (Table 2).

Although isolation of Bartonella henselae from peripheral blood of cats presenting with bacteremia was successful, PCR performed in the same samples did not yield any amplification.

Seroconversion occurred and IgG titers were determined by IFI only in the inoculated animal (Table 1). The other cats showed only weak reactions (titers < 64), and were considered non reagents.

DISCUSSION

Feline susceptibility to B. henselae infection was demonstrated by the isolation of the infectious agent from peripheral blood of the experimentally inoculated animal, and from other animals kept in contact, thus confirming the epidemiological role of cats^{1,5,8,9,11,17,18}.

Bacteremia was observed two months after inoculation, more lately than reported by others^1,17,18,20. The route of inoculation, type of bacterial strain, size and mode of inoculum preparation may have influenced the development of infection^1,13,14. Apparently, infection occurs early and bacteremia lasts longer, when infected blood is inoculated¹³.

All cats presenting with bacteremia were asymptomatic suggesting, as already reported by others^5,14,20, a low pathogenicity of B. henselae in cats. Seroconversion and bacteremia occurred simultaneously in the inoculated animal, but not in the others. In another study⁶, titers < 100 had low positive predictive value for CSD diagnosis in man.

Although the rising and falling of antibody titers together with bacteremia suggests a relationship between bacteremia and production of antibodies, it does not mean that the host is protected against infection⁵. The highest antibody titers are found in cats with bacteremia, with predictive positive value for some^1,14, while others⁵ consider that there is no relationship between threshold antibody titers and bacteremia. However, antibody-mediated immunity may be a way to control bacteremia, reducing its duration and the transmission of disease¹⁴.

The absence of relationship between bacteremia and antibody titers may justify the transmission of infectious agent from the inoculated animal to the others as demonstrated by B. henselae isolation, although significant production of antibodies did not occur (titers < 64). These findings are in accordance with those of CHOMEL et al.⁵ who found that some animals presenting with bacteremia did not present detectable IgG titers. Besides, non-reagent felines may be those that are partially tolerant to oral infection by B. henselae^8,11, what may have happened to the animals that were not inoculated which probably acquired the infection by ingestion of contaminated fleas or their feces.

Low IFI specificity to differentiate among Bartonella species¹⁵, mainly with respect to B. henselae and B. quintana, is troublesome for interpretation of antibody titers in man. This fact underlines the need to characterize isolated species. Fortunately, felines infection by B. quintana has not been proved²⁰, and positive reactions, when B. quintana is used as the IFI antigen are interpreted as cross-reactions^16,19.

The three PCR protocols performed in the present study enabled characterization of the isolates as belonging to Bartonella sp (HSP and FTSZ protocols) and to the henselae species (BH-PCR protocol). However, the same technique applied to peripheral blood failed to produce positive amplifications in the same animals and samples in which bacteria were isolated by hemoculture. Low sensitivity of PCR has already been reported in the literature¹⁵ and may be explained by an insignificant bacteria load, by heterogeneous distribution of bacteria in blood aliquots, and finally by a lack of sensitivity of the amplification protocols (primers, DNA extraction of whole blood instead of possibly only red cells, cycle programming – time and temperatures, rounds of amplification).

Our results corroborate those of other studies in that serological tests (IFI) have limited value in the assessment of bacteremia in animals. Besides, IFI is predictive of bacteremia only when titers are high, although an adequate negative predictive value is found when antibodies are absent. Moreover, we observed that PCR is not useful to detect B. henselae DNA in peripheral blood of felines and in the identification of eventual carriers of B. henselae, at least with the protocols used. Isolation of the agent, using hemoculture and characterization of the isolate by PCR, is the most efficient method to detect and type Bartonella species.

RESUMO

Infecção experimental e transmissão horizontal de Bartonella henselae em gatos domésticos

Procurou-se verificar a possibilidade de transmissão horizontal de B. henselae em 5 felinos confinados, dentre os quais apenas um foi inoculado experimentalmente por via subcutânea (SC) com 10⁵ UFC. Todos os felinos apresentavam infestação por pulgas. Para a avaliação da infecção foram utilizados: isolamento bacteriano (hemocultura), detecção de DNA específico pela Reação em Cadeia da Polimerase (PCR), com os protocolos HSP, FTSZ e BH-PCR, e a pesquisa de anticorpos específicos por Imunofluorescência Indireta (IFI). Os protocolos da PCR foram também utilizados para a caracterização do isolado da hemocultura. A inoculação de B. henselae resultou na infecção assintomática do animal inoculado, comprovada através da soroconversão e de bacteremia de 4 meses de duração, com o isolamento da bactéria na hemocultura. Todos os animais contactantes apresentaram bacteremia 6 meses após a data de inoculação experimental. No entanto, não apresentaram reação de IFI positiva. Em nenhum momento foi possível detectar DNA de B. henselae no sangue circulante, com as PCR utilizadas. Não obstante, a PCR possibilitou a identificação da bactéria isolada como sendo do gênero Bartonella (HSP e FTSZ-PCR) e espécie henselae (BH-PCR). Conclui-se que o isolamento bacteriano por meio da hemocultura constitui-se no método mais eficiente para a identificação dos felinos infectados e bacterêmicos. Estes resultados também evidenciam a possibilidade do papel das pulgas na transmissão de B. henselae em gatos.

ACKNOWLEDGMENTS

To Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) for the financial support. To Prof. Nílsson Roberti Benites, for the pictures.

Received: 09 March 2001

Accepted: 04 September 2001

Correspondence to: Marcelo de Souza Zanutto, Rua Manoel Joaquim Pera 85, 05539-030 São Paulo, SP, Brasil. E-mail: [e-mail] or [e-mail]

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(1

) Departamento de Clínica Médica da Faculdade de Medicina Veterinária e Zootecnia da Universidade de São Paulo, São Paulo, SP, Brasil.

(2

) Laboratório de Microbiologia do Departamento de Análises Clínicas da Faculdade de Ciências Farmacêuticas da Universidade de São Paulo, São Paulo, SP, Brasil.

(3

) Laboratório de Investigação Médica (LIM36) da Faculdade de Medicina, da Universidade de São Paulo, São Paulo, SP, Brasil.

Publication Dates

• Publication in this collection
  31 Oct 2001
• Date of issue
  Oct 2001

History

• Accepted
  04 Sept 2001
• Received
  09 Mar 2001