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Communication

Repeated Detection of Bartonella DNA in Feline Placenta: Potential Implications for Placental and Fetal Development

by
Charlotte O. Moore
1,
Ricardo Maggi
1,
Kelli Ferris
2 and
Edward B. Breitschwerdt
1,*
1
Intracellular Pathogens Research Laboratory, Department of Clinical Sciences, College of Veterinary Medicine, North Carolina State University, Raleigh, NC 27607, USA
2
Department of Clinical Sciences, College of Veterinary Medicine, North Carolina State University, Raleigh, NC 27607, USA
*
Author to whom correspondence should be addressed.
Animals 2025, 15(14), 2041; https://doi.org/10.3390/ani15142041
Submission received: 15 May 2025 / Revised: 27 June 2025 / Accepted: 4 July 2025 / Published: 11 July 2025
(This article belongs to the Section Companion Animals)
Versions Notes

Simple Summary

Three Bartonella species commonly infect the domestic cat, and may make it difficult for cats to become pregnant or have healthy offspring. The transmission of Bartonella from cats to kittens does not occur in healthy cats, but free-roaming domestic cats are often chronically stressed and/or co-infected with other pathogens. Therefore, we tested the placenta, fetus, ovary, and uterus tissue of free-roaming domestic cats to detect Bartonella DNA or culture live Bartonella. Bartonella DNA was detected in 28% (5/18) cats. Bartonella was not grown from any cat, but Bartonella DNA was detected in liquid culture. Therefore, we concluded that viable Bartonella may infect the cat’s placenta. Further research is necessary to understand if Bartonella can infect the placenta or be transmitted from cats to kittens.

Abstract

The domestic cat is the primary reservoir host of three flea-borne Bartonella species, one of which (Bartonella henselae) causes reduced fertility and reproductive failure in experimentally infected cats. Vertical transmission of Bartonella has been documented only in B-cell deficient mice, but not immunocompetent animals. As many free-roaming cats are chronically infected with Bartonella and may be immunocompromised by environmental stress or coinfection, we attempted to isolate Bartonella from the fetal and placental tissues of pregnant queens spayed during trap–neuter–release. Four samples from each tissue (ovary, uterus, fetus, and placenta) were split for direct DNA extraction, liquid culture, and culture on a blood agar plate. Samples from infected queens were inoculated into liquid media and sampled weekly for three weeks for DNA extraction and plating. Bartonella DNA was sequenced directly from 28% (5/18) of the free-roaming queens. For these five queens, liquid enrichment culture was attempted in duplicate for fetal and placental samples. Bartonella clarridgeiae DNA was amplified using qPCR liquid enrichment cultures from the placentas of two cats. These findings suggest that viable Bartonella organisms are present in feline reproductive tissue. Additional studies are needed to assess the transplacental transmission of Bartonella spp. and Bartonella’s influence on fetal development.

1. Introduction

The domestic cat is the primary reservoir host of three zoonotic Bartonella species (Bartonella clarridgeiae, Bartonella henselae, and Bartonella koehlerae) that are transmitted by the cat flea (Ctenocephalides felis). With a worldwide distribution, the cat and cat flea are ubiquitous in nearly all areas populated by human beings. Bartonella clarridgeiae is the most common species infecting cats and fleas, while B. henselae and B. koehlerae are more pathogenic in cats and humans [1,2]. Due to the difficulty of achieving a diagnosis of bartonellosis by culture isolation or PCR amplification of Bartonella spp. DNA, particularly in non-reservoir adapted hosts, studies involving naturally infected cats can provide insights into disease manifestations associated with infection. Bartonella DNA has been amplified from the female reproductive tissues of multiple species; however, its potential pathogenic effects remain unclear [3,4].
Current knowledge regarding the effect of Bartonella spp. infection on reproductive performance are based upon experimental infections and naturally infected animals or humans. Experimental infection of five queens with culture-grown B. henselae documented infertility upon repeated breeding attempts [5]. In the three cats that conceived, one kitten was born with hydrocephalus, and one was stillborn. In contrast, the uninfected control cat, bred by the same sire, became pregnant by the first breeding and delivered healthy kittens. Experimental infection of mice with Bartonella birtlesii, a species that infects mice and rats in nature, similarly documented increased fetal death and resorption and decreased fetal weight [6]. In mice experimentally infected with Bartonella taylorii, vertical transmission occurs only in the absence of B-cell adaptive immunity [7]. This finding in mice is reinforced by the lack of documented B. henselae vertical transmission in healthy, experimentally infected domestic cats [5,8]. These young, specific-pathogen-free cats, maintained in environmentally controlled facilities and feed an optimal diet, do not represent the health status of free-roaming cats with limited shelter and food. In addition, approximately 7% of feral cats are infected with FIV or FeLV, immunosuppressive retroviruses [9].
In the context of natural infection, multiple species of wild-caught voles (Microtus spp.) transmit Bartonella spp. to their offspring in a vector-free environment, with over half of offspring infected at 3 weeks of age [10]. Similar findings have been documented in smaller surveys of wild-caught rodents Peromyscus leucopus and Sigmodon hispidus [11]. Bartonella bovis infection in dairy cows was associated with a shorter interval from calving to artificial insemination and decreased incidence of placental retention, but other influences on bovine reproduction were not observed [12]. The limited associations of bartonellosis and reproduction to date supports the need to explore the effect of Bartonella spp. on reproduction in humans and other domestic and wildlife animal species. Prior documentation of B. henselae DNA in the placenta and fetal tissues of free-roaming cats suggests that the pathogen can circulate to the fetus [3]. To further assess the potential for placental or fetal Bartonella infection, we attempted to detect pathogen DNA via qPCR and to isolate Bartonella spp. from the fetus and placental tissues from naturally infected cats utilizing 21-day liquid enrichment culture and blood agar plating.

2. Materials and Methods

Reproductive tracts were obtained under NCSU Institutional Care and Use Committee protocol #21-468 via ovariohysterectomy from 18 free-roaming domestic cats during a trap-neuter-release program to assist in the control of free-roaming cat populations.
Reproductive tracts were frozen and transferred to the NCSU Intracellular Pathogens Research Laboratory for testing. Prior to dissection, tracts were thawed at 4 °C for two days. Tissue storage tubes were autoclaved. With a sterile scalpel, two ovary and two uterus samples were collected. After the scalpel and forceps were sterilized, the uterus was incised to reveal the placenta and amnionic sac, and scalpel and forceps were again sterilized. Placental samples were removed and the amnionic sac was lifted above a sterile Petri dish and cut to allow the fetus to drop into the dish. The scalpel and forceps were sterilized again. Total fetal length was measured crown to rump. Depending on the stage of gestation, fetal samples included the whole fetus or primarily abdominal organs. After sterilization of the scalpel and forceps, the procedure was repeated for the second fetus. All ovary, uterus, placental, and fetal samples were collected in duplicate.
Each tissue was individually bead beat (Fisherbrand™ Bead Mill 4 Mini Homogenizer, Fisher Scientific, Waltham, MA 02451, USA) with three metal beads until fully pulverized, and then inoculated with 1 mL of Brugge liquid enrichment culture media [13]. Bead beating controls were generated by bead-beating in an empty tube. Following Brugge inoculation, 200 µL was immediately removed for direct DNA extraction (Qaigen DNeasy Blood & Tissue Kit, Qiagen, Valencia, CA, USA) and Bartonella qPCR for the ssrA gene [14]. Fetal and placental samples from cats with ≥1 tissue qPCR positive for Bartonella DNA were selected for culture. For these samples, 200 µL of the original tissue and Brugge mixture was inoculated onto a blood agar plate (TSA with 5% sheep blood, ThermoFisher Scientific, Waltham, MA, USA). An additional 600 µL was inoculated into 10 mL of Brugge and 1 mL of defibrinated sheep’s blood (Carolina Biological Supply, Burlington, NC, USA). At 7, 14, and 21 days, 200 µL of the liquid enrichment culture was sampled for DNA extraction and qPCR, and 500 µL of liquid culture was inoculated onto a blood agar plate [13]. Blood agar plates and liquid culture were incubated at 37 °C and 5% CO2. Blood agar plates were kept for at least 6 weeks. Liquid culture negative controls consisted of Brugge culture medium subjected to bead-beating and liquid culture alone.
Based on the lack of successful blood agar plate isolation, frozen (−20 °C) stored tissues were utilized to reattempt culture isolation following incubation with Brugge (cat R4 and R7) or BAPGM (cat R9, R14, and R21) media [15]. BAPGM media culture was selected for R9, R14, and R21 to increase the likelihood of successful Bartonella isolation, as Brugge re-isolation of R4 and R7 had already been attempted and did not yield a Bartonella isolate. Tissues cultured in Brugge were performed as described above, while tissues cultured in BAPGM were inoculated into a T12.5 flask with 10 mL of BAPGM and 1 mL of defibrinated sheep’s blood.

3. Results

Reproductive tracts were obtained from 18 pregnant free-roaming cats. Bartonella spp. DNA was amplified from tissue extractions in five queens (5/18, 28%; Table 1). Bartonella DNA was amplified and sequenced from all sample types: ovary (11%, 4/36), placenta (8%, 3/36), fetus (2/36, 5.5%), and uterus (2/36, 5.5%). Three queens were infected with B. henselae, one was infected with Bartonella clarridgeiae, and one was infected with Bartonella vinsonii subsp. berkhoffii. Average fetal length (crown-rump) was 7.7 cm, but varied greatly with a minimum of 1.3 cm and maximum of 14.3 cm. Fetal length was similar in Bartonella DNA detected (7.17 ± 1.48 cm, mean ± SEM) and no Bartonella DNA detected (7.96 ± 0.98 cm) queens (p = 0.67).
Following liquid culture for 7, 14, and 21 days of incubation, Bartonella spp. DNA was amplified and sequenced from placental enrichment culture DNA extractions from two cats (Table 2). Infection with B. clarridgeiae was again documented using multiple enrichment culture timepoints in cat R7, in conjunction with co-infection with Bartonella koehlerae. Based upon direct tissue extraction and enrichment culture, cat R14 was co-infected with B. vinsonii subsp. berkhoffii and B. clarridgeiae.
Other fetal and placental enrichment culture samples were all qPCR-negative, and subculturing onto blood agar plates was not successful for any fetal or placental sample. Negative media and bead-beating controls remained qPCR-negative throughout the study period.

4. Discussion

This study documents the molecular presence of four Bartonella species in reproductive tissues of domestic free-roaming cats, including the detection of B. clarridgeiae DNA in ovary, uterus, fetus, and placental tissues. In addition, coinfection with two Bartonella spp. was documented in two out of five infected cats. As agar plate isolation was not successful for any tissue or at any enrichment subculture time point, documentation of Bartonella DNA in liquid culture supported bacterial growth, but did not definitively confirm bacterial viability. It is important to emphasize that even in tissues that are known to be Bartonella-infected, direct isolation or isolation with liquid subculture displays limited sensitivity. In R7 (a B. clarridgeiae infected cat), B. clarridgeiae was detected in all tissues using direct DNA extraction, as well as the placenta at all liquid culture days (7, 14, and 21 days); however, B. koehlerae coinfection was only detected in Brugge placental culture after 21 days of incubation. Bartonella clarridgeiae and B. henselae are detected in flea and cat samples more often than B. koehlerae, which, combined with our detection only at 21 days of liquid culture, suggests that diagnosis of B. koehlerae may be limited by low bacteremia or slower growth than other Bartonella species [1].
As with other mammalian species, the feline placenta is highly perfused with blood. Therefore, amplification of Bartonella spp. DNA may represent bacteria within the maternal blood supply or bacteria that have invaded and colonized placental tissues. As with the induction of placental lesions in mice experimentally infected with B. birtlesii, it is possible that Bartonella infection has a pathogenic effect on the placenta of the domestic cat [6]. Further research is necessary to determine if the reproductive failure of cats experimentally infected with B. henselae, as reported by Guptill and colleagues, may be attributed to pathological changes in placentation or fetal infection [5]. The effect of chronic bloodstream Bartonella infection on fetal weight, fetal resorption, and fetal developmental defects, the negative impacts of which were reported in B. birtlesii infected female mice, should also be explored in mothers, their progeny, and domestic and wildlife animal species [6]. Interestingly, Brucella, another alphaproteobacterial closely related to Bartonella, is a well-recognized cause of abortion and reproductive failure in multiple animal species [16].
The potential for vertical transmission of Bartonella spp. also has important implications for future disease manifestations in offspring and maintenance of these bacteria within various environments. Our inability to isolate Bartonella was expected, as Bartonella can be very difficult to isolate onto blood agar plates from chronically infected animal and human patients. The transition from liquid media (BAPGM or Brugge) to growth on a blood agar plate appears to be a rate-limiting step in the isolation process. In chronically infected cats or humans, Bartonella displays low organism numbers and a relapsing bacteremia, resulting in manuscripts utilizing multiple diagnostic methods to increase sensitivity [17,18,19]. The repeated documentation of B. clarridgeiae DNA at 7, 14, and 21 days of BAPGM enrichment cultures supports, but did not confirm, the presence of viable bacteria.
Free-roaming kittens display high mortality rates due to traumatic and infectious causes [20]. After experimental infection of B-cell deficient female mice, B. taylorii was repeatedly successfully isolated from offspring at 4–5 weeks [7]. The potential for stress, co-infection, chronic infection, or immunosuppression (e.g., retroviral infection, immunosuppressive drugs) to permit vertical transmission in mice, cats, or humans is unknown. The medical, microbiological, and pathological complexity of these and other factors illustrates the need for prospective, targeted, and structured studies to investigate the role of this genus in reproductive failures among animal species. Results that can be derived from studying Bartonella chronically infected free-roaming cats that are at high risk for FIV, FeLV, and other infections may provide a basis for comparative medical investigations.
In 2009, histologic, immunohistochemical, ultrastructural, and molecular methods were used to confirm B. henselae infection in an aborted equine fetus, providing the first evidence of reproductive failure in a non-reservoir host [21]. Case studies or case series also suggest in utero or perinatal transmission of Bartonella in humans. One case study documented perinatal transmission of B. henselae and B. vinsonii subsp. berkhoffii from the mother to her child, who died of a congenital heart defect at 9 days of age [4]. The twin brother and mother were B. henselae and B. vinsonii subsp. berkhoffii bacteremic when tested ten years after parturition, supporting the potential for chronic intravascular infection spanning a decade. As both B. henselae and B. vinsonii subsp. berkhoffii DNA were amplified and sequenced from the tissues of the child who died due to a congenital heart defect, a potential role for this genus should be investigated as a cause of abnormal embryogenesis [4]. In a small case series from Israel involving eight pregnant women diagnosed with Cat Scratch Disease, caused by B. henselae and potentially B. clarridgeiae, one woman had a spontaneous abortion, one terminated the pregnancy, and the remaining six had healthy newborns, with no reported sequalae for a median 4.5-year follow-up period [22]. Potential in utero infection with B. henselae was suspected in a 14-year-old boy with congenital cerebellar hypoplasia, who subsequently developed progressive, fluctuating neuropsychiatric symptoms [23]. Another case study reported the intrauterine fetal demise and assisted delivery at 31-week gestation from a 24-year-old mother presenting with relapsing low-grade fever and malaise for the previous month [24]. At this time, infection was not suspected and, therefore, additional microbiological investigation not pursued. Ultrasonography and computed tomography (CT) revealed a heteroechoic lesion in segment VI and VII of the liver in the mother. The mother subsequently underwent a right hepatectomy, which enabled the practitioners to discover an exophytic solid-cystic mass in segment VI and VII of the liver, a ruptured liver capsule, and intralesional hematoma, which were surgically resected. Histopathology, serology, and PCR results supported a diagnosis of B. henselae infection, with no prior reported cat contact.
The molecular detection of Bartonella coinfections in two queens only after testing more than one tissue using enrichment culture followed by DNA extraction and PCR testing further illustrates the limitations of current microbiological techniques for confirming or excluding infection with these bacteria. In a recent case report, infection with B. henselae was confirmed by enrichment brain biopsy culture in both BAPGM and Brugge media six years after the child was scratched on the face by a free-roaming cat [25]. Prior Bartonella spp. serology and enrichment culture of blood were both negative, illustrating the low-level chronic infection induced by these bacteria and the importance of tissue specific analyses in assessing whether an individual is or is not infected. Co-infection with multiple Bartonella species or other protozoa and bacterial species is widely reported in multiple animal species, including cats, dogs, and humans [3,26,27,28].

5. Conclusions

In conclusion, we documented the presence of Bartonella spp. DNA in ovary, uterus, placenta, and fetal tissues obtained from pregnant domestic free-roaming cats. Repeated enrichment culture of B. clarridgeiae from placental tissue of one cat supported the possibility of intrauterine infection with viable bacteria. Exploring the pathology of Bartonella infection in cat uterus, placental, and fetal tissues may identify previously unrecognized manifestations, with implications for zoonotic infection or transmission. The potential for vertical transmission in chronically infected mammalian reservoir and non-reservoir species remains unclear, and it is likely influenced by the infecting Bartonella spp., the evolutionary adaptation of the reservoir host, and the immune competence of the incidental host.

Author Contributions

Conceptualization, C.O.M., R.M., and E.B.B.; methodology, C.O.M., R.M., and E.B.B.; formal analysis, C.O.M.; investigation, C.O.M.; resources, K.F., R.M., and E.B.B.; data curation, C.O.M.; writing—original draft preparation, C.O.M.; writing—review and editing, C.O.M., K.F., R.M., and E.B.B.; supervision, K.F., R.M., and E.B.B.; project administration, K.F. and E.B.B.; funding acquisition, E.B.B. All authors have read and agreed to the published version of the manuscript.

Funding

This research was supported by donations to the Bartonella/Vector Borne Diseases Research Fund at the North Carolina State University College of Veterinary Medicine and NIH T34GM131947 training grant.

Institutional Review Board Statement

Animal samples were obtained under North Carolina State University Institutional Animal Care and Use Committee (IACUC) protocol #21-468.

Informed Consent Statement

Informed consent was obtained from all subjects involved in the study.

Data Availability Statement

The original contributions presented in this study are included in the article. Further inquiries can be directed to the corresponding author(s).

Acknowledgments

The authors would like to acknowledge the contributions of Katelyn Harris to sample dissection, culture, and pathogen detection. They would also like to thank the devoted trap–neuter–release volunteers and talented veterinarians, veterinary technicians, and veterinary students for collecting samples and working to reduce the free-roaming cat population around North Carolina.

Conflicts of Interest

In conjunction with S. Sontakke and North Carolina State University, E.B. Breitschwerdt holds US Patent No. 7,115,385 Media and Methods for Cultivation of Microorganisms, which was issued on 3 October 2006. He is a co-founder, shareholder, and Chief Scientific Officer for Galaxy Diagnostics, a company that provides advanced diagnostic testing for the detection of Bartonella spp. infections. Ricardo Maggi is a co-founder and the Chief Technical Officer for Galaxy Diagnostics Inc. All other authors declare that this research was conducted in the absence of any commercial or financial relationships that could be construed as potential conflicts of interest.

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Table 1. PCR amplification of the Bartonella ssrA gene from direct reproductive tissue extractions.
Table 1. PCR amplification of the Bartonella ssrA gene from direct reproductive tissue extractions.
Queen NumberFetal Length (cm)OvaryUterusPlacentaFetus
R471/2—B. henselae
(MK298190.1, 164/165 bp)		0/20/20/2
R711.51/2—B. clarridgeiae
(MK298175.1, 159/159 bp)		1/2—B. clarridgeiae
(MK298175.1, 159/159 bp)		1/2—B. clarridgeiae
(93/93, MN809215.1)		1/2—B. clarridgeiae
(MK298175.1, 159/159 bp)
R96.50/21/2—B. henselae
(MK298190.1, 156/157 bp)		0/20/2
R142.41/2—B. vinsonii subsp. berkhoffii
(MG432827.1, 149/150)		0/20/20/2
R218.51/2—B. henselae
(MK298190.1, 158/158 bp)		0/20/20/2
Number of qPCR-positive samples from each tissue and Bartonella spp. determined using NCBI BLAST comparison (blast.ncbi.nlm.nih.gov, accessed on 4 July 2025). Negative samples were omitted for readability. Fetal length is expressed as the average of two fetuses in centimeters.
Table 2. Detection of Bartonella DNA via qPCR amplification and sequencing from a liquid culture medium at multiple time points.
Table 2. Detection of Bartonella DNA via qPCR amplification and sequencing from a liquid culture medium at multiple time points.
Queen NumberTissueCulture MediumCulture DayBartonella spp.
R7PlacentaBrugge (first culture)7B. clarridgeiae
(MK298175.1, 159/159 bp)
R7PlacentaBrugge (first culture)14B. clarridgeiae
(MK298175.1, 159/159 bp)
R7PlacentaBrugge (first culture)21B. clarridgeiae
(MK298175.1, 159/159 bp)
R7PlacentaBrugge (second culture)21B. koehlerae
(JN029769.1, 166/167 bp)
R14PlacentaBrugge (second culture)14B. clarridgeiae
(MK298175.1, 165/168 bp)
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MDPI and ACS Style

Moore, C.O.; Maggi, R.; Ferris, K.; Breitschwerdt, E.B. Repeated Detection of Bartonella DNA in Feline Placenta: Potential Implications for Placental and Fetal Development. Animals 2025, 15, 2041. https://doi.org/10.3390/ani15142041

AMA Style

Moore CO, Maggi R, Ferris K, Breitschwerdt EB. Repeated Detection of Bartonella DNA in Feline Placenta: Potential Implications for Placental and Fetal Development. Animals. 2025; 15(14):2041. https://doi.org/10.3390/ani15142041

Chicago/Turabian Style

Moore, Charlotte O., Ricardo Maggi, Kelli Ferris, and Edward B. Breitschwerdt. 2025. "Repeated Detection of Bartonella DNA in Feline Placenta: Potential Implications for Placental and Fetal Development" Animals 15, no. 14: 2041. https://doi.org/10.3390/ani15142041

APA Style

Moore, C. O., Maggi, R., Ferris, K., & Breitschwerdt, E. B. (2025). Repeated Detection of Bartonella DNA in Feline Placenta: Potential Implications for Placental and Fetal Development. Animals, 15(14), 2041. https://doi.org/10.3390/ani15142041

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