Open Veterinary Journal, (2025), Vol. 15(12): 6480-6486

Research Article

10.5455/OVJ.2025.v15.i12.32

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Molecular detection of Mycoplasma haemofelis in cats infested with Ctenocephalides felis in Padang, West Sumatra, Indonesia

Hesti Rahayu¹, Teguh Rianda¹, Dwi Priyowidodo^2*, Irkham Widiyono³, Vika Ichsania Ninditya²

¹Postgraduate Student, Master of Veterinary Science Program, Faculty of Veterinary Medicine, Gadjah Mada University, Yogyakarta, Indonesia

²Department of Parasitology, Faculty of Veterinary Medicine, Universitas Gadjah Mada, Yogyakarta, Indonesia

³Department of Internal Medicine, Faculty of Veterinary Medicine, Universitas Gadjah Mada, Yogyakarta, Indonesia

*Corresponding Author: Dwi Priyowidodo. Department of Parasitology, Faculty of Veterinary Medicine, Universitas Gadjah Mada, Yogyakarta, Indonesia. Email: priyo [at] ugm.ac.id

Submitted:28/07/2025 Revised: 22/10/2025 Accepted: 03/11/2025 Published: 31/12/2025

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Abstract

Background: Mycoplasma haemofelis infection is considered an important feline disease and is suspected to be transmitted via the ectoparasitic vector Ctenocephalides felis, particularly in tropical areas.

Aim: This study aimed to detect M. haemofelis at the molecular level in cats naturally infested with C. felis in Padang City, West Sumatra, Indonesia.

Methods: A total of 61 cats were examined to assess their clinical condition and flea infestation levels, followed by blood sampling for polymerase chain reaction analysis targeting the 16S rRNA gene of M. haemofelis.

Results: The PCR results revealed a high prevalence of infection (72.13%; 44/61). The level of flea infestation was positively associated with the frequency of M. haemofelis infection, with rates of 40.0%, 60.0%, and 83.3% in mild, moderate, and severe infestations, respectively. Statistical analysis revealed a significant association (p < 0.05) between flea infestation level and infection status. Cats kept in multi-cat households were more frequently infected than those kept in single-cat households. No significant associations were found between infection occurrence and age, sex, or maintenance pattern.

Conclusion: A high prevalence of M. haemofelis was detected in cats infested with C. felis in Padang City, with a significant association with flea infestation level and housing pattern.

Keywords: Mycoplasma haemofelis, Ctenocephalides felis, Cat, PCR, Padang.

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Introduction

Arthropod-borne vector-borne diseases represent a global health issue because they involve various important pathogens, including bacteria, viruses, and protozoa. Ctenocephalides felis is one of the arthropod vectors that play a significant role in transmitting infectious agents in animals (Otranto et al., 2025; Páez-Triana et al., 2025). This species is recognized as a primary vector for Bartonella henselae and Rickettsia felis, both of which are important zoonotic pathogens that can cause disease in humans and animals (Moore et al., 2024a). Ctenocephalides felis is a hematophagous ectoparasite belonging to the family Pulicidae and is widely distributed worldwide. Heavy infestations in cats can result in clinical manifestations, such as flea allergic dermatitis, secondary skin lesions due to scratching, opportunistic infections, and physiological stress that can lead to immunosuppression (Pereira et al., 2025). In addition to its role as an ectoparasite, C. felis also harbors the pathogenic bacterium Mycoplasma haemofelis (Zarea et al., 2023; Razgūnaitė et al., 2024).

Mycoplasma haemofelis is a hemotropic bacterium belonging to the genus Mycoplasma, previously known as Haemobartonella felis. This bacterium lacks a cell wall, making it relatively resistant to various antimicrobial agents and capable of surviving under diverse environmental conditions (Cortés Sánchez, 2022). Morphologically, M. haemofelis adheres to the outer surface of erythrocytes without penetrating the cell membrane and is recognized as the most pathogenic hemoplasma species in cats (Yasmin et al., 2022). The clinical manifestations of M. haemofelis infection in cats can range from subclinical to severe hemolytic anemia, particularly in cats with compromised immune systems, which are often accompanied by lethargy, pale mucous membranes, anorexia, and weight loss (Lima, 2025).

Although the transmission routes of M. haemofelis are not yet fully understood, several mechanisms are suspected to be involved, including blood transfusion, aggressive interactions between individuals, and the role of ectoparasite vectors, such as C. felis (Tasker et al., 2018; Pimpjong et al., 2025; Shi et al., 2025). Although M. haemofelis DNA was detected within fleas in recent molecular studies, its prevalence was low, and some results may represent nonspecific amplification (Moore et al., 2024b). The detection of M. haemofelis DNA in fleas further supports its potential role as a mechanical vector for the transmission of this pathogen (Mifsud et al., 2020; Razgūnaitė et al., 2024).

Flea infestations tend to increase in warm and humid tropical environments, accelerating the life cycle of C. felis and increasing the risk of pathogen transmission (Pereira et al., 2025). Padang City, which has a tropical climate with an average temperature of 25 °C and humidity of 80% (BPS (Statistics Indonesia), 2024), provides an optimal environment for C. felis development, potentially increasing the risk of M. haemofelis transmission among the local cat population.

To date, no data are available regarding the occurrence of M. haemofelis infection in cats infested with C. felis in Padang City. Therefore, this study aimed to molecularly detect M. haemofelis in cats infested with C. felis in Padang, West Sumatra, Indonesia. The results of this study are expected to contribute to vector-borne disease control efforts and to improve overall animal health.

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Materials and Methods

This study included 61 cats naturally infested with C. felis, originating from Padang City, West Sumatra. Each cat underwent a thorough physical examination to assess its clinical condition and determine the degree of flea infestation. The study population comprised 57 owned cats and 4 cats from local shelters. Ctenocephalides felis infestation was assessed using the thumb counting method, in which fleas were counted through visual inspection within 3 minutes by manually parting the hair at predetermined predilection sites, including the dorsal midline, inguinal region, axillae, flanks, and tail base (Fox et al., 1969; Gregory et al., 1995). According to Marchiondo et al. (2007), the infestation level was classified as mild, moderate, or severe based on the total number of fleas found on the animal’s body. The flea infestation levels based on flea counts are presented in Table 1.

Table 1. Levels of flea infestation (Marchiondo et al., 2007).

Blood samples (0.5 ml) were collected from the cephalic vein of each cat in ethylenediaminetetraacetic acid (EDTA) tubes following the standard procedures described by Reynolds et al. (2007). Genomic DNA was extracted using the Genomic DNA Mini Kit (Blood/Cultured Cell) (Geneaid Biotech Ltd., Taiwan) according to the manufacturer’s instructions. The extracted DNA was used as a template for polymerase chain reaction (PCR), employing specific primers designed based on the 16S rRNA gene sequence of H. felis from GenBank accession number U95297.1, yielding an amplicon of approximately 590 bp. PCR amplification consisted of an initial denaturation at 95°C for 5 minutes, followed by 30 cycles of denaturation at 94°C for 1 minutes, annealing at 56°C for 30 seconds, extension at 72°C for 30 seconds, and a final extension at 72°C for 10 minutes. PCR was performed using a Biorad PTC Tempo Thermal Cycler, California, USA. The PCR products were electrophoresed on a 1.5% agarose gel and visualized using a UV transilluminator (UVP, Upland, CA, USA). This protocol follows previously published molecular detection methods for M. haemofelis (Imre et al., 2020).

Statistical analysis

The data obtained were analyzed descriptively to assess flea infestation levels and the frequency of cats infected with M. haemofelis based on polymerase chain reaction results. Distribution analysis between PCR-positive and PCR-negative cats for M. haemofelis was performed using a chi-square χ² test, considering flea infestation level C. felis sex, age, housing system, and number of cats per household. In addition, logistic regression analysis was performed to evaluate the strength of association between these risk factors and M. haemofelis infection. All analyses were conducted at a 95% confidence level (p<0.05). Using the IBM SPSS Statistics software version 26.0.

Ethical approval

This study was approved by the Ethics Committee of the Faculty of Veterinary Medicine, Universitas Gadjah Mada, Yogyakarta, Indonesia approval number: 39/EC-FKH/int./2025).

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Results

This study included 61 cats naturally infested with C. felis. The 16S rRNA gene of M. haemofelis was detected in 44 of 61 blood samples (72.13%) using conventional PCR. Figure 1 shows the results of the conventional PCR electrophoresis for M. haemofelis.

Fig. 1. Conventional PCR electrophoresis on 1.5% agarose gel.

The distribution of flea infestation levels was predominant in the severe category (59.0%; 36/61), followed by the moderate (32.8%; 20/61) and mild (8.2%; 5/61) categories. Accordingly, the prevalence of M. haemofelis infection increased progressively in the mild (40%; 2/5), moderate (60%; 12/20), and severe (83.3%; 30/36) categories. No infections were observed in cats without flea infestation (Table 2).

Table 2. Frequency of M. haemofelis infection based on flea infestation levels (n=61).

Analysis of the general characteristics showed that the samples included flea infestation level, various age groups, sexes, housing systems, and household cat populations, which were categorized as single-cat and multi-cat households. Table 3 presents the association analysis between M. haemofelis incidence and sample characteristics. Cats with severe infestation had higher odds of infection than those with mild infestation (OR=7.50, 95% CI: 1.02–54.99, p=0.047). Sex, age, and housing system were not significantly associated with M. haemofelis infection with the occurrence of infection (p > 0.05). In contrast, the household cat population showed a tendency toward increased risk, with cats from many-cat households having higher odds than those from few-cat households (OR=9.21, 95% CI: 0.89–95.86, p=0.063).

Table 3. Logistic regression analysis of characteristics of cats associated with M. haemofelis infection.

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Discussion

The present study revealed a high prevalence (72.13%) of M. haemofelis infection among cats naturally infested with C. felis. This prevalence is considerably higher than that in China (52.5%) (Shi et al., 2025), Romania (23.7%) (Imre et al., 2020), the Czech Republic (12.7%) (Lobo et al., 2021), Brazil (8.4%) (Melo TB de et al., 2023), Iran (2.2%) (Hoseinpoor et al., 2024), and Türkiye (1.74%) (Altay et al., 2025). The high prevalence observed in this study is most likely influenced by homogeneous population characteristics, as all sampled cats were infested with C. felis, which are suspected to act as potential vectors for M. haemofelis (Abdullah et al., 2019).

A significant association was observed between the severity of flea infestation and M. haemofelis infection (p=0.043). The infection rate increased from 40% in mild infestations to 83.3% in severe infestations. This pattern may be associated with the tropical humid climate of Padang City, which is characterized by high rainfall and relatively stable humidity throughout the year, which supports the survival of C. felis and increases the risk of infestation in cats (Pereira et al., 2025).

A notable finding of this study was that cats with severe flea infestations were more likely to be infected with M. haemofelis than those with mild infestations (OR=7.50, 95% CI: 1.02–54.99, p=0.047). These results support the concept that a higher ectoparasite burden elevates the likelihood of hemotropic pathogen transmission (Altay et al., 2025; Herrera León, 2025) and are consistent with previous studies highlighting the role of fleas as mechanical vectors of M. haemofelis (Abdullah et al., 2019; Mifsud et al., 2020; Razgūnaitė et al., 2024). Berzina et al. (2021) reported that most infected cats did not receive routine antiparasitic treatment. Lappin (2018) noted that heavy infestations can cause skin lesions and immunosuppression, thereby increasing the risk of secondary infections. Although flea-mediated transmission is possible, its efficiency is low (Woods et al., 2005). While direct blood transmission is the primary route, flea infestation remains an important indicator of infection risk (Tasker et al., 2018).

No significant associations were found between infection and sex, age, or housing system of cats. This finding is consistent with the report by Berzina et al. (2021) but differs from other studies that reported an increased risk of aggressive behavior in male cats (Pekel and Duru, 2022; Herrera León, 2025) and adult cats (Lobová et al., 2021; Lima, 2025). Interestingly, cats living in multi-cat households showed a tendency toward increased (OR=9.21, 95% CI: 0.89–95.86, p=0.063), possibly due to higher ectoparasite infestation and pathogen transmission potential in dense populations (Tasker et al., 2018; Kamyingkird et al., 2021).

Studies on M. haemofelis infection in Indonesia remain limited and are generally restricted to individual case reports without population-based incidence data. Several reports have included cases in Bandung (Satriawan and Octaviani, 2021), two cases in Bali (Tjokorda Istri Agung Pradnya Dewi Pemayun et al., 2024; Billa et al., 2024), and a case of hypoproteinemia in Yogyakarta (Jayanti et al., 2023). Most of these reports relied on microscopic examination of blood smears for diagnosis, which has limited sensitivity and specificity compared with molecular methods. Therefore, the use of 16S rRNA gene-based PCR in this study provides significant diagnostic advantages and serves as an initial contribution to population-based prevalence data on cats infested with C. felis in Indonesia.

The diagnostic accuracy strongly depends on the method used. Several studies have demonstrated that PCR has significant advantages over blood smear examination. Sagare et al. (2024) reported a prevalence of M. haemofelis of 5.4% using blood smears, which increased to 13.5% when PCR was used. In Brazil, a difference was observed between conventional PCR (15.6%) and quantitative PCR (28.6%) (Herrera León, 2025). Therefore, molecular methods are essential tools for detecting subclinical cases that are often missed by microscopic examinations (Cortés Sanchez, 2022).

The prevalence of M. haemofelis infection exceeding 72.1% in cats infested with C. felis highlights the importance of ectoparasite control and routine molecular screening, even in animals without clinical signs. These findings support the need for population-based preventive strategies and environmental management to control M. haemofelis infections. The cross-sectional design did not allow for assessing the causal relationship between flea infestation and M. haemofelis infection. The identification focused solely on one hemoplasma species without detecting possible coinfections with Candidatus M. haemominutum or Candidatus M. turicensis. Immunological status, presence of other viruses such as FIV, FeLV, and environmental factors were not further analyzed due to data limitations. Longitudinal studies and multigenus molecular approaches are highly recommended to improve our understanding of hemoplasma epidemiology and transmission dynamics.

Despite these limitations, this study provides the first molecular evidence of M. haemofelis infection in naturally infected cats with C. felis in Indonesia. Previous national reports were restricted to microscopic findings based on individual cases (Satriawan and Octaviani, 2021; Billa et al., 2024), whereas molecular data have been lacking. Therefore, the present findings offer important baseline information for Southeast Asia and emphasize the role of ectoparasite control in tropical regions, consistent with earlier detection of M. haemofelis DNA in cat fleas (Abdullah et al., 2019).

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Conclusion

This study detected a high prevalence of M. haemofelis in cats infested with C. felis in Padang City. A significant association was observed between infection status and flea infestation level and housing pattern.

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Acknowledgments

The authors extend their sincere gratitude to the Master of Veterinary Science Program, Faculty of Veterinary Medicine, Universitas Gadjah Mada, Yogyakarta, and the Department of Parasitology and Internal Medicine for academic guidance and support. Appreciation is also given to the Provincial Animal Hospital of West Sumatra, Balai Veteriner Bukittinggi, Paw’s Vet Padang, Platinum Vet Care, Aurora Pet Care, and Dunia Pet Care for their valuable assistance and for providing access to clinical cases and sample collection during this study.

Funding

This study was self-funded by the authors.

Authors' contributions

Hesti Rahayu: Original draft writing, conceptualization, data curation, formal analysis, and visualization. Teguh Rianda: conceptualization, funding acquisition, supervision, and project administration. Dwi Priyowidodo: investigation, methodology, supervision, and validation. Irkham Widiyono: Data curation, investigation, supervision, and validation. Vika Ichsania Ninditya: Preparation of the manuscript’s initial draft.

Conflict of interest

The authors have no conflicts of interest to declare.

Data availability

All references are open access, so data can be obtained from the internet.

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