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Introduction the Prevalence of Bartonella, Hemoplasma, And Article The prevalence of Bartonella, hemoplasma, and Rickettsia felis infections in domestic cats and in cat fleas in Ontario Ali Kamrani, Valeria R. Parreira, Janice Greenwood, John F. Prescott Abstract The prevalence of persistent bacteremic Bartonella spp. and hemoplasma infections was determined in healthy pet cats in Ontario. Blood samples from healthy cats sent to a diagnostic laboratory for routine health assessment over the course of 1 y were tested for Bartonella spp. using both polymerase chain reaction (PCR) and blood culture, and for the presence of hemoplasma by PCR. The overall prevalence of Bartonella spp. by PCR and by culture combined was 4.3% (28/646) [3.7% (24/646) Bartonella henselae, 0.6% (4/646) Bartonella clarridgeiae]. The novel B. henselae PCR developed for this study demonstrated nearly twice the sensitivity of bacterial isolation. The overall prevalence of hemoplasma was 4% (30/742) [3.3% (25/742) Candidatus Mycoplasma haemominutum, 0.7% (5/742) Mycoplasma haemofelis]. There was no significant difference between the prevalence of infection by season or by age (# 2 y, . 2 y). Candidatus Mycoplasma turicensis was identified, for the first time in Canada, in 1 cat. The prevalence of Bartonella (58%) and hemoplasma (47% M. haemofelis, 13% M. haemominutum) in blood from a small sampling (n = 45) of stray cats was considerably higher than that found in healthy pet cats. The prevalence of Rickettsia felis in cat fleas was also assessed. A pool of fleas from each of 50 flea-infested cats was analyzed for the presence of R. felis by PCR. Rickettsia felis was confirmed, for the first time in Canada, in 9 of the 50 samples. Therefore, the prevalence of Bartonella and hemoplasma infection in healthy pet cats is relatively low. Further, the control of cat fleas is important because of the public health significance of Bartonella and R. felis infection. Résumé La prévalence d’une bactériémie persistante à Bartonella spp. et d’une infection à hémoplasma a été déterminée dans une population de chats en santé provenant de l’Ontario. Des échantillons sanguins de chat en santé envoyés à un laboratoire de diagnostic pour un bilan de santé de routine durant une période d’une année ont été éprouvés pour Bartonella spp. par réaction d’amplification en chaîne par la polymérase (PCR) et par hémoculture, et pour la présence d’hémoplasma par PCR. La prévalence globale de Bartonella spp. par PCR et culture combinés était de 4,3 % (28/646) [3,7 % (24/646 Bartonella henselae, 0,6 % (4/646) B. clarridgeiae]. L’épreuve PCR développée pour la présente étude avait une sensibilité presque le double de l’isolement bactérien. La prévalence globale d’hémoplasma était de 4 % (30/742) [3,3 % (25/742) Candidatus Mycoplasma haemominutum, 0,7 % (5/742) M. haemofelis]. Il n’y avait pas de différence significative entre les prévalences d’infection selon la saison ou le groupe d’âge (# 2 ans, . 2 ans). Candidatus Mycoplasma turicensis a été identifié chez un chat pour la première fois au Canada. La prévalence de Bartonella (58 %) et d’hémoplasma (47 % M. haemofelis, 13 % M. haemominutum) dans le sang d’un échantillonnage restreint (n = 45) de chats errants était considérablement plus élevée que chez des chats en santé. La prévalence de Rickettsia felis chez des puces de chats a également été évaluée. Un pool de puces provenant de chacun de 50 chats infestés par des puces a été analysé par PCR pour la présence de R. felis. La présence de R. felis a été confirmée pour la première fois au Canada dans 9 des 50 échantillons. Ainsi, la prévalence d’infection par Bartonella et hémoplasma chez des chats en santé est relativement faible. De plus, la maîtrise des puces de chat est importante compte tenu de l’impact sérieux en santé publique des infections par Bartonella et R. felis. (Traduit par Docteur Serge Messier) Introduction B. henselae, B. clarridgeiae, and B. kohlerae (2–4), all of which may cause various clinical manifestations in humans (4,5). Bartonella henselae is Healthy cats may be subclinical carriers of blood-borne bacterial the major causative agent of cat scratch disease (CSD) in immuno- infections that are potentially serious zoonotic pathogens and which competent people and of bacillary angiomatosis and hepatic peliosis may cause severe diseases in other cats. The genus Bartonella is in patients with a defective immune system, especially those infected comprised of fastidious hemotropic bacteria that have been increas- with human immunodeficiency virus (HIV) (5,6). As a result of the ingly identified in a wide range of domestic and wild animals (1). development of sensitive molecular techniques for the diagnosis of Domestic cats are the primary reservoir of 3 species of Bartonella, Bartonella organisms in human patient tissue samples, and because Department of Pathobiology, and Centre for Public Health and Zoonoses, University of Guelph, Guelph, Ontario N1G 2W1 (Kamrani, Parreira, Prescott); Vita-Tech Laboratories, 1345 Denison Street, Markham, Ontario L3R 5V2 (Greenwood). Address all correspondence to Dr. J.F. Prescott; telephone: (519) 824-4120, ext 54453; fax: (519) 824-5930; e-mail: [email protected] Received September 6, 2007. Accepted January 8, 2008. 2008;72:411–419 The Canadian Journal of Veterinary Research 411 of the increasing numbers of patients with defective immune sys- kit (QIAamp DNA Blood Mini Kit; Qiagen, Mississauga, Ontario). tems through HIV infection or the use of immunosuppressive drugs, Purified DNA was eluted in 150 mL elution buffer and stored at Bartonella spp. have emerged as agents of zoonotic infections (7). This 220°C. Pooled flea samples were prepared for DNA extraction by study is the first to use both blood culture and polymerase chain first rinsing in sterile distilled water for 5 min at room temperature. reaction (PCR) to investigate the prevalence of Bartonella bacteremia Each sample was macerated completely in a 1.5 mL microcentrifuge in both pet and stray domestic cats in Ontario. tube using a sterile pipette tip. The DNA was extracted, according to Feline hemobartonellosis is a bacteremic infection in cats that may the manufacturer’s instructions, using the QIAamp DNA Blood Mini be associated with severe hemolytic anemia. The etiologic agents of Kit (Qiagen), eluted in 100 mL elution buffer and stored at 220°C. To this infection, Mycoplasma haemofelis and Mycoplasma haemominutum, monitor the extraction process for any foreign DNA contamination, may cause severe hemolytic anemia, and subtle anemia or subclinical a negative “DNA extraction” control, consisting of 200 mL sterile infection, respectively, in cats (8). Candidatus Mycoplasma turicensis phosphate buffered saline (pH 7.2, PBS), was included in each batch is a recently recognized hemotropic mycoplasma, first identified of samples processed. in 2005 in Switzerland in a cat with hemolytic anemia (9). This Various primer combinations, each targeting the 16S–23S rRNA study is the first to investigate the prevalence of the hemoplasmas, intergenic region of the genus Bartonella, were assessed for their including Candidatus Mycoplasma turicensis, in pet and stray cats amplification efficiency of plasmid DNA containing the target in Ontario. gene of B. henselae (ATCC 4988). Oligonucleotide sequences of each Rickettsia felis is a recently recognized agent of flea-borne spotted primer are listed in Table I. The following primer combinations fever in humans (10–12). The cat flea is the major vector of this organ- were examined: primer pair JF 311/JR 473, as described by Jensen ism. In endemic areas of the United States, peri-urban opossums are et al (15); primer pair MF 321/MR 983, as described by Maggi the major reservoir host of R. felis. The reservoir potential of cats has and Breitschwerdt (16); and, a novel combination of the primers yet to be determined, although experimental infection of cats with MF 321/JR 473. The PCR amplifications were performed in a 25 mL R. felis has been demonstrated (13,14). One objective of this study reaction mix containing a ready-to-use PCR buffer that included a was to determine if R. felis is present in cat fleas in Canada. proof-reading hot start DNA polymerase (Platinum PCR SuperMix High Fidelity; Invitrogen, Burlington, Ontario), 0.3 pmol/mL of each primer, and 2 mL DNA template (0.02 ng, plasmid DNA containing Materials and methods B. henselae target gene). Use of proof-reading DNA polymerase on Bartonella DNA when samples were extracted from blood, removed Blood samples some nonspecific amplification observed with Taq polymerase (data Blood from healthy domestic cats in southern Ontario was col- not shown). Amplification conditions used in this assay consisted of lected in tubes containing ehtylenediamine tetra-acetic acid (EDTA) denaturation at 94°C for 2 min, 45 cycles of 30 s of denaturation at anticoagulant, and submitted to a laboratory (Vita-Tech Laboratories, 94°C, 30 s of annealing at 58°C, 30 s of extension at 68°C, followed Markham, Ontario) as part of a routine “wellness” program for by an additional cycle of 7 min extension at 68°C. The predicted size cats. Care was taken to exclude samples from animals that were of the amplicons resulting from this PCR reaction were calculated either suspect or clinically ill. A convenience sampling method was to be 152 bp, 136 bp, 233 bp, 242 bp, and 187 bp for B. henselae, used, whereby approximately 45 cats . 2 years old, and 14–45 cats, B. clarridgeiae, B. elizabethae, B. vinsonii subsp. berkhoffii, and B. bovis, # 2 years old, were collected each month. A total of 742 samples respectively. The PCR amplification products were visualized by were collected over a period of 12 mo (April 2006 to March 2007). ethidium bromide fluorescence after electrophoresis in a 3% agarose Blood samples were also collected from 45 stray cats, found within gel.
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