Bartonellae in Domestic and Stray Cats from Israel: Comparison of Bacterial Cultures and High-Resolution Melt Real-Time PCR As Diagnostic Methods

Abstract

To determine the occurrence of feline bartonellosis in Israel, blood samples were collected from 179 stray and 155 domestic cats from 18 cities or villages in central and northcentral Israel. Samples were screened for Bartonella infection by culture isolation and molecular detection using high-resolution melt (HRM) real-time PCR assay targeting the 16S-23S rRNA internal transcribed spacer (ITS). All positive samples were confirmed by two additional HRM real-time PCR assays targeting two fragments of the β-subunit of RNA polymerase (rpoB) and the 16S rRNA genes. The prevalence of Bartonella spp. infection in the general tested population was 25.1% (84/334). A higher prevalence was detected in the stray (30.7%; 55/179) than the domestic cats (18.7%; 29/155). Bartonella henselae, Bartonella clarridgeiae, and Bartonella koehlerae were highly prevalent in both cat populations, however their distribution among the two populations varied significantly (p=0.016). B. clarridgeiae and B. koehlerae were found to be more prevalent in stray than domestic cats, whereas B. henselae was evenly distributed. Co-infection with two or more different Bartonella spp. was determined in 2.1% (7) of the cats. The ITS HRM real-time PCR assay used in this study was shown to have a greater screening power than bacterial isolation, detecting 94.0% (79/84) compared to 35.7% (30/84), respectively, of all positive samples. The high prevalence of these zoonotic Bartonella species, coupled with the overpopulation of stray cats, and increased numbers of domestic cats in the major urban centers in Israel represent a significant threat for the public health in this country.

Relationship between the Presence of Bartonella Species and Bacterial Loads in Cats and Cat Fleas (Ctenocephalides felis) under Natural Conditions

“…In a recent experimental-infection study, Chomel et al (33) have shown that the ability to produce bacteremia in cats varies among nonfeline-associated Bartonella species. In a previous study performed by our group, a different rodent Bartonella genotype was detected in a cat (24), suggesting that cats may be infected with other rodent-associated strains. Thus, the biological role of C. felis fleas in the transmission of nonfeline bartonellae to cats and other mammalian hosts (including humans) needs to be further investigated.…”

“…Genomic DNA samples obtained from Bartonella-free cat blood and Bartonella-free fleas were used as negative controls, and ultrapure water was used as a nontemplate control (NTC). The expected HRM profiles of Bartonella species ranged from 80 to 85°C (24); thus, melt profiles out of this range were considered nonspecific products.…”

“…The latter studies have based the infection status and quantification of bacterial loads on bacterial isolation (11,18). However, it has been shown that isolationbased methods underestimate the rates of Bartonella species infection in cats in comparison to molecular-analysis-based assays (24). Thus, investigation of the distribution of Bartonella species and their infection loads in both hosts and vectors through a molecular-analysis-based quantitative approach was warranted.…”

Identification of Different Bartonella Species in the Cattle Tail Louse (Haematopinus quadripertusus) and in Cattle Blood

“…The occurrence of B. henselae in cattle may reflect the frequent spread of this species in Israel. Recently, the prevalence of this species was determined in cats from this country and shown to be evenly distributed in stray and domestic cats (30). Thus, despite their acknowledged feline association, B. henselae seems to have a permissive cycle in nature since it has been detected in several ecological niches (hosts and vectors) (18,41).…”

“…Thermal conditions were performed according to the authors' recommendations. In order to increase the sensitivity of the Bartonella screening, HRM real-time PCR assays were performed for the amplification of partial fragments for the ITS (190 bp) and the transfermRNA (ssrA) gene (301 bp) using primer sets and protocols as previously described (29)(30)(31). The real-time PCRs were carried out in a 20-l final volume containing 1 l of 0.5 M solution of each primer, 0.6 l of 1.5 M solution of Syto9 (Invitrogen, CA), 3.4 l of DDW, 10 l of MAXIMA Hot-Start PCR master mix 2X (Thermo Scientific, Surrey, United Kingdom), and 4 l of each genomic DNA.…”