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1.  Birds as potential reservoirs of tick-borne pathogens: first evidence of bacteraemia with Rickettsia helvetica 
Parasites & Vectors  2014;7:128.
Background
Birds have long been known as carriers of ticks, but data from the literature are lacking on their role as a reservoir in the epidemiology of certain tick-borne disease-causing agents. Therefore, the aim of this study was to evaluate the presence of three emerging, zoonotic tick-borne pathogens in blood samples and ticks of birds and to assess the impact of feeding location preference and migration distance of bird species on their tick infestation.
Methods
Blood samples and ticks of birds were analysed with TaqMan real-time PCRs and conventional PCR followed by sequencing.
Results
During the spring and autumn bird migrations, 128 blood samples and 140 ticks (Ixodes ricinus, Haemaphysalis concinna and a Hyalomma specimen) were collected from birds belonging to 16 species. The prevalence of tick infestation and the presence of tick species were related to the feeding and migration habits of avian hosts. Birds were shown to be bacteraemic with Rickettsia helvetica and Anaplasma phagocytophilum, but not with Candidatus Neoehrlichia mikurensis. The prevalence of rickettsiae was high (51.4%) in ticks, suggesting that some of them may have acquired their infection from their avian host.
Conclusion
Based on the present results birds are potential reservoirs of both I. ricinus transmitted zoonotic pathogens, R. helvetica and A. phagocytophilum, but their epidemiological role appears to be less important concerning the latter, at least in Central Europe.
doi:10.1186/1756-3305-7-128
PMCID: PMC3976504  PMID: 24679245
Ground feeding birds; Migratory birds; Ticks; Rickettsia helvetica; Anaplasma phagocytophilum; Candidatus Neoehrlichia mikurensis
2.  Prevalence of Francisella tularensis and Francisella-Like Endosymbionts in the Tick Population of Hungary and the Genetic Variability of Francisella-Like Agents 
Abstract
Several new taxa belonging to the genus Francisella have been described recently. The present study describes the prevalence of Francisella tularensis and Francisella-like endosymbionts (FLE) in ticks collected from Hungary from 2007 to 2009 and characterizes the genetic variability of FLEs. A total of 5402 Ixodid ticks (Ixodes ricinus, I. acuminatus, Dermacentor marginatus, D. reticulatus, Haemaphysalis inermis, H. concinna, H. punctata) were collected from vegetation and animal hosts and tested with conventional PCR, detecting the 16S rRNA and tul4 genes. F. tularensis ssp. holarctica was found in 2 pools of H. concinna and 1 pool of D. reticulatus, both representing minimum prevalence (calculated with 1 infected tick per pool) of 0.27% whereas the sequences of a FLE were detected in 11 pools of D. reticulatus showing a minimum prevalence of 3%. Although the tul4 gene sequence of this FLE was identical to all Hungarian and Portuguese FLEs found earlier, and the 16S rRNA sequence was also identical to the sequence of the endosymbiont of D. reticulatus described in Bulgaria, these 16S rRNA gene coding sequences differed in 2 nucleotides from the one found earlier in this tick species in Hungary. This divergence may appear to be a minor difference between the sequences, potentially even resulting from a technical failure, but it could also indicate a significant difference stemming from the conservative genetic character of Francisellaceae. Thus, it raises a question about the number of FLE variants circulating in D. reticulatus in Europe and indicates the need for further data about the FLEs described in other parts of the continent and new FLE genotyping markers.
doi:10.1089/vbz.2012.1065
PMCID: PMC3601718  PMID: 23421891
Francisella-like endosymbiont; Francisella tularensis; Tick; Zoonosis
3.  Bovine besnoitiosis emerging in Central-Eastern Europe, Hungary 
Parasites & Vectors  2014;7:20.
Background
Besnoitia besnoiti, the cause of bovine besnoitiosis, is a cyst-forming coccidian parasite that has recently been shown to be spreading in several Western and Southern European countries.
Findings
Clinical cases of bovine besnoitiosis were confirmed for the first time in Hungary, by histological, serological and PCR analyses.
Conclusions
This is the first report of autochthonous bovine besnoitiosis in Central-Eastern Europe. The emergence of bovine besnoitiosis in this region represents a further example, when human activity (i.e. cattle trading) is the main factor involved in the geographical spread of an infectious disease.
doi:10.1186/1756-3305-7-20
PMCID: PMC3895772  PMID: 24410743
Besnoitia besnoiti; Cattle; Mechanical vector; Vector-borne; Emerging disease
4.  First evidence of Candidatus Neoehrlichia mikurensis in Hungary 
Parasites & Vectors  2013;6:267.
Altogether 2004 Ixodes ricinus ticks, from 37 places in Hungary, were analysed in pools with a recently developed multiplex real-time PCR for the presence of Candidatus Neoehrlichia mikurensis and for other representatives of the genus. Ca. Neoehrlichia mikurensis was identified in nine sampling sites, indicating three separated endemic regions along the borders of Hungary. In addition, results of samples from seven places (except for the western part of the country) were positive in the genus-specific (Ca. Neoehrlichia sp.) PCR, but were negative for Ca. Neoehrlichia mikurensis.
doi:10.1186/1756-3305-6-267
PMCID: PMC3849741  PMID: 24341500
Tick-borne diseases; Zoonosis; Epidemiology
5.  GAPDH Pseudogenes and the Quantification of Feline Genomic DNA Equivalents 
Quantitative real-time PCR (qPCR) is broadly used to detect and quantify nucleic acid targets. In order to determine cell copy number and genome equivalents, a suitable reference gene that is present in a defined number in the genome is needed, preferably as a single copy gene. For most organisms, a variable number of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) pseudogenes have been reported. However, it has been suggested that a single-copy of the GAPDH pseudogene is present in the feline genome and that a GAPDH assay can therefore be used to quantify feline genomic DNA (gDNA). The aim of this study was to determine whether one or more GAPDH pseudogenes are present in the feline genome and to provide a suitable alternative qPCR system for the quantification of feline cell copy number and genome equivalents. Bioinformatics and sequencing results revealed that not just one but several closely related GAPDH-like sequences were present in the cat genome. We thus identified, developed, optimized, and validated an alternative reference gene assay using feline albumin (fALB). Our data emphasize the need for an alternative reference gene, apart from the GAPDH pseudogene, for the normalization of gDNA levels. We recommend using the fALB qPCR assay for future studies.
doi:10.1155/2013/587680
PMCID: PMC3655645  PMID: 23738070
6.  Genome Sequence for “Candidatus Mycoplasma haemominutum,” a Low-Pathogenicity Hemoplasma Species 
Journal of Bacteriology  2012;194(4):905-906.
We present the genome sequence of “Candidatus Mycoplasma haemominutum” strain Birmingham 1, a low-pathogenicity feline hemoplasma strain.
doi:10.1128/JB.06560-11
PMCID: PMC3272962  PMID: 22275103
7.  Stimulation with a class A CpG oligonucleotide enhances resistance to infection with feline viruses from five different families 
Veterinary Research  2012;43(1):60.
Domestic cats are commonly affected by viral pathogens that induce lengthy infections with fatal outcomes. Prevention of viral propagation is of primordial importance in shelters and catteries, where cats from different backgrounds have narrow contacts. Oligonucleotides (ODN) containing cytosine-phosphate-guanosine motifs of class A (CpG-A) are highly potent synthetic inducers of innate antiviral mechanisms. The aim of this study was to test their ability to modulate innate immune responses and prevent viral replication as stand-alone agents in the domestic cat. CpG-A stimulation of feline peripheral blood mononuclear cells (PBMCs) enhanced their proliferation, increased the presence of co-stimulatory molecules on their surface and influenced their gene expression profiles in an antiviral orientation. Incubation of the supernatants of CpG-A stimulated PBMCs with feline cell lines of epithelial and fibroblastic origin induced expression of the antiviral myxovirus resistance (Mx) gene in these target cells, which also showed enhanced resistance to feline viruses from five distinct families, namely Coronaviridae, Herpesviridae, Caliciviridae, Parvoviridae, and Retroviridae. Most importantly, subcutaneous administration of CpG-A in domestic cats systemically increased the expression of Mx, reaching maximal levels within 24 h. Plasma from treated cats could furthermore inhibit viral replication in vitro. Altogether, our data highlight the promising potential of CpG-A to induce a preventive antiviral state in the cat and to protect feline populations against a broad range of virus infections.
doi:10.1186/1297-9716-43-60
PMCID: PMC3537549  PMID: 22906110
8.  Protection from reinfection in “Candidatus Mycoplasma turicensis”-infected cats and characterization of the immune response 
Veterinary Research  2012;43(1):82.
“Candidatus Mycoplasma turicensis” (CMt) is a hemoplasma species of felids. Recent evidence has shown that cats that overcome bacteremia may be protected from reinfection. The purposes of this study were to (1) re-inoculate ostensibly recovered cats, (2) evaluate the immune response and (3) assess CMt tissue loads. Fifteen specified pathogen-free cats were subcutaneously inoculated with CMt: 10 cats (group A) had previously undergone bacteremia and recovered, and 5 naïve cats (group B) served as controls. CMt infections were monitored by real-time PCR using blood and tissue, and the humoral immune response was assessed using DnaK ELISA. Cytokine mRNA expression levels were measured by real-time PCR, and lymphocyte subsets were detected by flow cytometry. The cats in group A were protected from reinfection (no detectable bacteremia) and showed a transient decrease in antibodies. Eosinophilia was noted in cats from group A. The cats from group B became PCR-positive and seroconverted. All of the tissues analyzed from the cats in group B but none of the tissues analyzed from the cats in group A were CMt PCR-positive. Significant changes were observed in the expression of tumor necrosis factor-α, interferon-γ, interleukin-4 and the Th2/Th1 ratio in both groups. The cats from group A occasionally showed higher numbers of CD4+, CD8+, CD4+CD25+ and CD5+MHCII+ T lymphocytes than the control cats. In conclusion, this study describes, for the first time, the occurrence of immunological protection within the same hemoplasma species. Furthermore, the immune response during CMt infections appeared to be skewed toward the Th2 type.
doi:10.1186/1297-9716-43-82
PMCID: PMC3558367  PMID: 23216686
9.  First morphological characterization of ‘Candidatus Mycoplasma turicensis’ using electron microscopy 
Veterinary Microbiology  2011;149(3-4):367-373.
At least three haemotropic mycoplasmas have been recognized in cats: Mycoplasma haemofelis (Mhf), ‘Candidatus Mycoplasma haemominutum’ (CMhm) and ‘Candidatus M. turicensis’ (CMt). The latter was originally identified in a Swiss pet cat with haemolytic anaemia and shown to be prevalent in domestic cats and wild felids worldwide using molecular methods. So far, there has been no confirmatory morphological evidence of the existence of CMt presumably due to low blood loads during infection while CMhm has only been characterized by light microscopy with discrepant results. This study aimed to provide for the first time electron microscopic characteristics of CMt and CMhm and to compare them to Mhf. Blood samples from cats experimentally infected with CMt, CMhm and Mhf were used to determine copy numbers in blood by real-time PCR and for transmission and scanning electron microscopy. High resolution scanning electron microscopy revealed CMt and CMhm to be discoid-shaped organisms of 0.3 μm in diameter attached to red blood cells (RBCs). In transmission electron microscopy of CMt, an oval organism of about 0.25 μm with several intracellular electron dense structures was identified close to the surface of a RBC. CMhm and CMt exhibited similar morphology to Mhf but had a smaller diameter. This is the first study to provide morphological evidence of CMt thereby confirming its status as a distinct haemoplasma species, and to present electron microscopic features of CMhm.
doi:10.1016/j.vetmic.2010.11.020
PMCID: PMC3127424  PMID: 21183295
‘Candidatus Mycoplasma turicensis’; ‘Candidatus Mycoplasma haemominutum’; Haemoplasma; Haemotropic Mycoplasma; Electron microscopy; Real-time PCR
10.  Identification, Molecular Characterization, and Occurrence of Two Bovine Hemoplasma Species in Swiss Cattle and Development of Real-Time TaqMan Quantitative PCR Assays for Diagnosis of Bovine Hemoplasma Infections▿  
Journal of Clinical Microbiology  2010;48(10):3563-3568.
Concomitantly with an outbreak of fatal anaplasmosis in a cattle herd in Switzerland in 2002, we detected two bovine hemoplasma species in diseased animals: Mycoplasma wenyonii (formerly Eperythrozoon wenyonii) and a second, novel bovine hemoplasma species later designated “Candidatus Mycoplasma haemobos” (synonym, “Candidatus Mycoplasma haemobovis”). The second species was characterized by a shorter 16S rRNA gene. The aims of the present study were to provide a detailed molecular characterization of this species, to develop specific quantitative real-time PCR assays for the two bovine hemoplasma species, and to apply these assays in order to evaluate the prevalence and clinical significance of the hemoplasmas. Sequencing of the near-complete 16S rRNA gene of the second hemoplasma revealed that it was 94% identical to that of Mycoplasma haemofelis, an anemia-inducing feline hemoplasma species, but less than 85% identical to that of the bovine hemoplasma M. wenyonii. Using the newly developed assays, a total of 159 animals from the anaplasmosis outbreak were reexamined. In addition, we tested 57 clinically ill and 61 healthy Swiss cattle, as well as 47 calves. Both hemoplasmas were highly prevalent in adult cattle but occurred rarely in calves. Animals from the herd with the fatal anemia outbreak were more frequently infected with M. wenyonii and exhibited higher M. wenyonii blood loads than animals with unrelated diseases and healthy animals. Coinfections may increase the pathogenicity and clinical significance of bovine hemoplasmosis.
doi:10.1128/JCM.02224-09
PMCID: PMC2953077  PMID: 20686093
11.  Exposure of cats to low doses of FeLV: seroconversion as the sole parameter of infection 
Veterinary Research  2009;41(2):17.
In felids, feline leukemia virus (FeLV) infection results in a variety of outcomes that range from abortive (virus readily eliminated and never detectable) to progressive infection (persistent viremia and viral shedding). Recently, a novel outcome was postulated for low FeLV infectious doses. Naïve cats exposed to faeces of persistently infected cats seroconverted, indicating infection, but remained negative for provirus and p27 antigen in blood. FeLV provirus was found in some tissues but not in the bone marrow, infection of which is usually considered a necessary stage for disease progression. To investigate the impact of low FeLV doses on young cats and to test the hypothesis that low dose exposure may lead to an unknown pathogenesis of infection without involvement of the bone marrow, 21 cats were infected oronasally with variable viral doses. Blood p27, proviral and viral loads were followed until week 20 post-infection. Tissue proviral loads were determined as well. The immune response was monitored by measuring FeLV whole virus and p45 antibodies; and feline oncornavirus-associated cell membrane antigen (FOCMA) assay. One cat showed regressive infection (transient antigenemia, persistent provirus-positivity, and seroconversion) with provirus only found in some organs at sacrifice. In 7 of the 20 remaining cats FOCMA assay positivity was the only sign of infection, while all other tests were negative. Overall, the results show that FeLV low dose exposure can result in seroconversion during a presumed abortive infection. Therefore, commonly used detection methods do not detect all FeLV-infected animals, possibly leading to an underestimation of the prevalence of infection.
doi:10.1051/vetres/2009065
PMCID: PMC2789331  PMID: 19861115
FeLV; pathogenesis; infection outcome; abortive infection; FOCMA assay
12.  Detection of feline haemoplasma species in experimental infections by in-situ hybridisation 
Microbial Pathogenesis  2011;50(2):94-99.
The aim of this study was to use fluorescence in-situ hybridisation (FISH) to search for the tissues and cell types important in survival and persistence of Mycoplasma haemofelis, “Candidatus Mycoplasma haemominutum” or “Candidatus Mycoplasma turicensis” in infected cats. A 16S rDNA probe for each species was applied to formalin-fixed, paraffin wax-embedded tissues sections collected from experimentally infected cats.
Tissues (n = 12) were collected, at necropsy, from ten cats which had been infected with M. haemofelis, and one each with “Ca. M. haemominutum” and “Ca. M. turicensis”. M. haemofelis specific hybridisation was present on red blood cells (RBCs) in all tissues from acutely infected cats, but not the majority of tissues from chronically infected cats. “Ca. M. haemominutum” specific hybridisation was present on scattered RBCs within the spleen and liver. Specific probe hybridisation was not detected in any of the “Ca. M. turicensis” infected tissues.
Haemoplasmas were detected on the surface of RBCs only and not any other cell type. Additionally, FISH was limited by sensitivity and could not detect the lower numbers of organisms present in tissues of cats chronically infected with M. haemofelis. Occasional organisms were detected in cats acutely infected with “Ca. M. haemominutum” but not “Ca. M. turicensis”.
doi:10.1016/j.micpath.2010.09.003
PMCID: PMC3032890  PMID: 21129480
Haemoplasma; Mycoplasma haemofelis; “Candidatus Mycoplasma haemominutum”; “Candidatus Mycoplasma turicensis”; Fluorescence in-situ hybridisation
13.  Chronic "Candidatus Mycoplasma turicensis" infection 
Veterinary Research  2011;42(1):59.
"Candidatus Mycoplasma turicensis" infects felids. The pathogenesis of "Candidatus M. turicensis" chronic infection is poorly understood. The goals of the present study were to (1) induce reactivation of the infection in chronic carrier cats by attempted immunosuppression, (2) identify potential tissue sequestration using real-time TaqMan® PCR and (3) monitor the humoral immune response by DnaK enzyme-linked immunosorbent assay (ELISA). Ten specified pathogen-free cats that had ostensibly recovered from experimental "Candidatus M. turicensis" infection were used: five cats (group 1) received high dose methylprednisolone (attempted immunosuppression), while five cats served as untreated controls (group 2). Besides weekly blood samples, tissue samples were collected from bone marrow, kidney, liver and salivary glands at selected time points. The cats in group 1 had significantly lower lymphocyte counts and higher blood glucose levels after methylprednisolone administration than the controls. After methylprednisolone administration one blood and three tissue samples from cats in group 1 tested PCR-positive; before the administration, only one sample was positive. All other samples tested PCR-negative. All cats stayed seropositive; the antibody levels of the cats in group 1 showed a significant transient decrease after methylprednisolone administration. This is the first study to report the presence of "Candidatus M. turicensis" in tissues of chronically infected cats and the persistence of anti-feline hemoplasma antibodies in the absence of detectable bacteremia. Methylprednisolone administration did not lead to a significant reactivation of the infection. Our results enhance the knowledge of "Candidatus M. turicensis" infection pathogenesis and are clinically relevant to the prognosis of hemoplasma-infected cats.
doi:10.1186/1297-9716-42-59
PMCID: PMC3090992  PMID: 21507220
14.  Detection of Humoral Response Using a Recombinant Heat Shock Protein 70, DnaK, of Mycoplasma haemofelis in Experimentally and Naturally Hemoplasma-Infected Cats▿ †  
Clinical and Vaccine Immunology : CVI  2010;17(12):1926-1932.
Hemoplasmas is the trivial name given to a group of erythrocyte-parasitizing bacteria of the genus Mycoplasma. Of the feline hemoplasmas, Mycoplasma haemofelis is the most pathogenic, while “Candidatus Mycoplasma haemominutum” and “Candidatus Mycoplasma turicensis” are less pathogenic. Shotgun libraries of fragmented M. haemofelis genomic DNA were constructed, and random colonies were selected for DNA sequencing. In silico-translated amino acid sequences of putative open reading frames were compared to mass spectrometry data from M. haemofelis protein spots identified as being immunogenic by two-dimensional gel electrophoresis and Western blotting. Three of the spots matched the predicted sequences of a heat shock protein 70 (DnaK) homolog, elongation factor Ts, and a fragment of phosphoglycerate kinase found during library screening. A full-length copy of the M. haemofelis dnaK gene was cloned into Escherichia coli and recombinantly expressed. Recombinant M. haemofelis DnaK was purified and then used in Western blotting and an enzyme-linked immunosorbent assay (ELISA) to investigate the humoral immune response during acute infection in cats experimentally infected with M. haemofelis, “Ca. Mycoplasma haemominutum,” or “Ca. Mycoplasma turicensis”. The recombinant M. haemofelis DnaK ELISA also was used to screen clinical samples submitted for hemoplasma PCR testing to a commercial laboratory (n = 254). Experimentally infected cats became seropositive following infection, with a greater and earlier antibody response seen in cats inoculated with M. haemofelis than those seen in cats inoculated with “Ca. Mycoplasma haemominutum” or “Ca. Mycoplasma turicensis,” by both Western blotting and ELISA. Of the clinical samples, 31.1% had antibodies detected by the ELISA but only 9.8% were positive by PCR for one or more hemoplasmas.
doi:10.1128/CVI.00320-10
PMCID: PMC3008206  PMID: 20926695
15.  Identification, Characterization, and Application of a Recombinant Antigen for the Serological Investigation of Feline Hemotropic Mycoplasma Infections ▿ ‡  
Clinical and Vaccine Immunology : CVI  2010;17(12):1917-1925.
In felids, three hemotropic mycoplasma species (hemoplasmas) have been described: Mycoplasma haemofelis, “Candidatus Mycoplasma haemominutum,” and “Candidatus Mycoplasma turicensis.” In particular, M. haemofelis may cause severe, potentially life-threatening hemolytic anemia. No routine serological assays for feline hemoplasma infections are available. Thus, the goal of our project was to identify and characterize an M. haemofelis antigen (DnaK) that subsequently could be applied as a recombinant antigen in a serological assay. The gene sequence of this protein was determined using consensus primers and blood samples from two naturally M. haemofelis-infected Swiss pet cats, an experimentally M. haemofelis-infected specific-pathogen-free cat, and a naturally M. haemofelis-infected Iberian lynx (Lynx pardinus). The M. haemofelis DnaK gene sequence showed the highest identity to an analogous protein of a porcine hemoplasma (72%). M. haemofelis DnaK was expressed recombinantly in an Escherichia coli DnaK knockout strain and purified using Ni affinity, size-exclusion, and anion-exchange chromatography. It then was biochemically and functionally characterized and showed characteristics typical for DnaKs (secondary structure profile, thermal denaturation, ATPase activity, and DnaK complementation). Moreover, its immunogenicity was assessed using serum samples from experimentally hemoplasma-infected cats. In Western blotting or enzyme-linked immunosorbent assays, it was recognized by sera from cats infected with M. haemofelis, “Ca. Mycoplasma haemominutum,” and “Ca. Mycoplasma turicensis,” respectively, but not from uninfected cats. This is the first description of a full-length purified recombinant feline hemoplasma antigen that can readily be applied in future pathogenesis studies and may have potential for application in a diagnostic serological test.
doi:10.1128/CVI.00282-10
PMCID: PMC3008192  PMID: 20876820
16.  In vivo transmission studies of ‘Candidatus Mycoplasma turicensis’ in the domestic cat 
Veterinary Research  2009;40(5):45.
The natural transmission routes of the three feline haemotropic mycoplasmas – Mycoplasma haemofelis, ‘Candidatus Mycoplasma haemominutum’, and ‘Candidatus Mycoplasma turicensis’ (CMt) – are largely unknown. Since CMt has been detected in the saliva of infected cats using PCR, we hypothesised that direct transmission via social or aggressive contact may occur. The aim of this study was to evaluate this transmission route. CMt-positive saliva and blood samples were obtained from three prednisolone-treated specific pathogen-free (SPF) cats that were infected intraperitoneally with CMt. Five SPF cats were inoculated with CMt-positive saliva or blood subcutaneously to mimic cat bites, and five cats were inoculated orally with blood or oronasally with saliva to mimic social contact. Blood samples were monitored for CMt infection using quantitative real-time PCR and for seroconversion using a novel western blot assay. Neither oronasal nor subcutaneous inoculation with CMt-positive saliva led to CMt infection in the recipient cats, as determined by PCR, independent of prior prednisolone treatment. However, when blood containing the same CMt dose was given subcutaneously, 4 of the 5 cats became PCR-positive, while none of the 5 cats inoculated orally with up to 500 μL of CMt-positive blood became PCR-positive. Subsequently, the latter cats were successfully subcutaneously infected with blood. All 13 CMt-exposed cats seroconverted. In conclusion, CMt transmission by social contact seems less likely than transmission by aggressive interaction. The latter transmission may occur if the recipient cat is exposed to blood from an infected cat.
doi:10.1051/vetres/2009028
PMCID: PMC2701178  PMID: 19505421
haemotropic mycoplasma; transmission; ‘Candidatus Mycoplasma turicensis’; real-time TaqMan PCR; seroconversion
17.  Chimeric Feline Coronaviruses That Encode Type II Spike Protein on Type I Genetic Background Display Accelerated Viral Growth and Altered Receptor Usage▿  
Journal of Virology  2009;84(3):1326-1333.
Persistent infection of domestic cats with feline coronaviruses (FCoVs) can lead to a highly lethal, immunopathological disease termed feline infectious peritonitis (FIP). Interestingly, there are two serotypes, type I and type II FCoVs, that can cause both persistent infection and FIP, even though their main determinant of host cell tropism, the spike (S) protein, is of different phylogeny and displays limited sequence identity. In cell culture, however, there are apparent differences. Type II FCoVs can be propagated to high titers by employing feline aminopeptidase N (fAPN) as a cellular receptor, whereas the propagation of type I FCoVs is usually difficult, and the involvement of fAPN as a receptor is controversial. In this study we have analyzed the phenotypes of recombinant FCoVs that are based on the genetic background of type I FCoV strain Black but encode the type II FCoV strain 79-1146 S protein. Our data demonstrate that recombinant FCoVs expressing a type II FCoV S protein acquire the ability to efficiently use fAPN for host cell entry and corroborate the notion that type I FCoVs use another main host cell receptor. We also observed that recombinant FCoVs display a large-plaque phenotype and, unexpectedly, accelerated growth kinetics indistinguishable from that of type II FCoV strain 79-1146. Thus, the main phenotypic differences for type I and type II FCoVs in cell culture, namely, the growth kinetics and the efficient usage of fAPN as a cellular receptor, can be attributed solely to the FCoV S protein.
doi:10.1128/JVI.01568-09
PMCID: PMC2812337  PMID: 19906918
18.  Development and Application of a Universal Hemoplasma Screening Assay Based on the SYBR Green PCR Principle▿  
Journal of Clinical Microbiology  2009;47(12):4049-4054.
Hemotropic mycoplasmas (hemoplasmas) are the causative agents of infectious anemia in several mammalian species. Their zoonotic potential has recently been substantiated by the identification of a feline hemoplasma isolate in an immunocompromised human patient. Although species-specific diagnostic molecular methods have been developed, their application as screening tools is limited due to the species diversity of hemoplasmas. The goals of this study were to develop a universal hemoplasma screening assay with broad specificity based on the SYBR green PCR principle, to compare the assay with hemoplasma-specific TaqMan PCR, and to analyze potential tick vectors and human blood samples to address the zoonotic potential. The newly developed PCR assay based on the 16S rRNA gene amplified feline, canine, bovine, porcine, camelid, and murine hemoplasmas, as well as Mycoplasma penetrans and Mycoplasma pneumoniae. The lower detection limit for feline and canine hemoplasmas was 1 to 10 copies/PCR. The assay exhibited 98.2% diagnostic sensitivity and 92.1% diagnostic specificity for feline hemoplasmas. All 1,950 Ixodes ticks were PCR negative, suggesting that Ixodes ticks are not relevant vectors for the above-mentioned hemoplasma species in Switzerland. None of the 414 blood samples derived from anemic or immunocompromised human patients revealed a clear positive result. The SYBR green PCR assay described here is a suitable tool to screen for known and so-far-undiscovered hemoplasma species. Positive results should be confirmed by specific TaqMan PCR or sequencing.
doi:10.1128/JCM.01478-09
PMCID: PMC2786680  PMID: 19828748
19.  Dominance of highly divergent feline leukemia virus A progeny variants in a cat with recurrent viremia and fatal lymphoma 
Retrovirology  2010;7:14.
Background
In a cat that had ostensibly recovered from feline leukemia virus (FeLV) infection, we observed the reappearance of the virus and the development of fatal lymphoma 8.5 years after the initial experimental exposure to FeLV-A/Glasgow-1. The goals of the present study were to investigate this FeLV reoccurrence and molecularly characterize the progeny viruses.
Results
The FeLV reoccurrence was detected by the presence of FeLV antigen and RNA in the blood and saliva. The cat was feline immunodeficiency virus positive and showed CD4+ T-cell depletion, severe leukopenia, anemia and a multicentric monoclonal B-cell lymphoma. FeLV-A, but not -B or -C, was detectable. Sequencing of the envelope gene revealed three FeLV variants that were highly divergent from the virus that was originally inoculated (89-91% identity to FeLV-A/Glasgow-1). In the long terminal repeat 31 point mutations, some previously described in cats with lymphomas, were detected. The FeLV variant tissue provirus and viral RNA loads were significantly higher than the FeLV-A/Glasgow-1 loads. Moreover, the variant loads were significantly higher in lymphoma positive compared to lymphoma negative tissues. An increase in the variant provirus blood load was observed at the time of FeLV reoccurrence.
Conclusions
Our results demonstrate that ostensibly recovered FeLV provirus-positive cats may act as a source of infection following FeLV reactivation. The virus variants that had largely replaced the inoculation strain had unusually heavily mutated envelopes. The mutations may have led to increased viral fitness and/or changed the mutagenic characteristics of the virus.
doi:10.1186/1742-4690-7-14
PMCID: PMC2837606  PMID: 20167134
20.  Quantitative TaqMan® real-time PCR assays for gene expression normalisation in feline tissues 
BMC Molecular Biology  2009;10:106.
Background
Gene expression analysis is an important tool in contemporary research, with real-time PCR as the method of choice for quantifying transcription levels. Co-analysis of suitable reference genes is crucial for accurate expression normalisation. Reference gene expression may vary, e.g., among species or tissues; thus, candidate genes must be tested prior to use in expression studies. The domestic cat is an important study subject in both medical research and veterinary medicine. The aim of the present study was to develop TaqMan® real-time PCR assays for eight potential reference genes and to test their applicability for feline samples, including blood, lymphoid, endocrine, and gastrointestinal tissues from healthy cats, and neoplastic tissues from FeLV-infected cats.
Results
RNA extraction from tissues was optimised for minimal genomic DNA (gDNA) contamination without use of a DNase treatment. Real-time PCR assays were established and optimised for v-abl Abelson murine leukaemia viral oncogene homolog (ABL), β-actin (ACTB), β-2-microglobulin (B2M), β-glucuronidase (GUSB), hydroxymethyl-bilane synthase (HMBS), hypoxanthine phosphoribosyltransferase (HPRT), ribosomal protein S7 (RPS7), and tryptophan 5-monooxygenase activation protein, zeta polypeptide (YWHAZ). The presence of pseudogenes was confirmed for four of the eight investigated genes (ACTB, HPRT, RPS7, and YWHAZ). The assays were tested together with previously developed TaqMan® assays for feline glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and the universal 18S rRNA gene. Significant differences were found among the expression levels of the ten candidate reference genes, with a ~106-fold expression difference between the most abundant (18S rRNA) and the least abundant genes (ABL, GUSB, and HMBS). The expression stability determined by the geNorm and NormFinder programs differed significantly. Using the ANOVA-based NormFinder program, RPS7 was the most stable gene in the tissues studied, followed by ACTB and ABL; B2M, HPRT, and the 18S rRNA genes were the least stable ones.
Conclusion
The reference gene expression stability varied considerably among the feline tissues investigated. No tested gene was optimal for normalisation in all tissues. For the majority of the tissues, two to three reference genes were necessary for accurate normalisation. The present study yields essential information on the correct choice of feline reference genes depending on the tissues analysed.
doi:10.1186/1471-2199-10-106
PMCID: PMC2803789  PMID: 20003366
21.  Distribution of Mycoplasma haemofelis in blood and tissues following experimental infection 
Microbial Pathogenesis  2009;47(6-2):334-340.
The aim of the study was to describe blood and tissue copy number distribution during Mycoplasma haemofelis infection and determine if sequestration of organisms in body tissues could explain blood copy number cycling in infected cats. Thirteen domestic–shorthaired cats were used. Blood samples were regularly collected, and at a differing time point post-infection for each cat, tissue samples also collected, for quantitative PCR (qPCR). Absolute haemoplasma copy numbers were calculated for all blood and tissue samples, as well as an estimation of the ratio of tissue haemoplasma copy number to that expected in the tissue if a positive qPCR result arose due to tissue blood supply alone. Cats with high or moderate M. haemofelis blood copy numbers at the time of tissue collection had fewer M. haemofelis copies in most tissues than expected due to the tissue blood supply alone; only splenic and lung tissues consistently contained more M. haemofelis. However tissues collected from cats at a time of very low M. haemofelis blood copy numbers, when putative copy number cycling nadirs were occurring, were usually qPCR negative. Hence no evidence of significant tissue M. haemofelis sequestration was found in this study to explain the copy number cycling reported with this feline haemoplasma species.
doi:10.1016/j.micpath.2009.09.009
PMCID: PMC2791849  PMID: 19782126
Haemoplasma; Quantitative real-time PCR; Sequestration, M. haemofelis
22.  Description of outcomes of experimental infection with feline haemoplasmas: Copy numbers, haematology, Coombs’ testing and blood glucose concentrations 
Veterinary Microbiology  2009;139(3-4):323-332.
The aim of this study was to compare blood copy, haematological and glucose values between cats experimentally infected with either Mycoplasma haemofelis (Group HF: 10 cats), ‘Candidatus M. haemominutum’ (Group HM: 3 cats) or ‘Candidatus M. turicensis’ (Group TU: 3 cats). Blood samples were collected regularly up to 85 days post-infection (DPI) for haemoplasma real-time quantitative PCR, haematology, Coombs’ testing and blood glucose measurement. Statistical analysis was performed using a general linear model (ANOVA) appropriate for a repeated measures experiment with significance set as P < 0.05. Cats in Group TU had significantly lower blood copy numbers than cats in Group HF (P < 0.001) and HM (P < 0.001). All Group HF cats developed anaemia (often severe), macrocytosis and evidence of erythrocyte-bound antibodies whereas Groups HM and TU cats did not. Group HF had significantly lower PCVs, haemoglobin concentrations and red blood cell counts, and significantly higher mean cell volumes, than Groups HM and TU. In Group HF, erythrocyte-bound antibodies reactive at 4 °C (both IgM and IgG) appeared between 8 and 22 DPI and persisted for two to four weeks, whereas those reactive at 37 °C (primarily IgG) appeared between 22 and 29 DPI and persisted for one to five weeks. In most cats antibodies appeared after the fall in haemoglobin started. Although Group TU had significantly lower glucose concentrations than Groups HF (P = 0.006) and HM (P = 0.027), mean blood glucose concentrations remained within the reference range in all groups. This study demonstrates that M. haemofelis infection, in contrast to ‘Candidatus M. haemominutum’ and ‘Candidatus M. turicensis’ infection, can result in a severe macrocytic anaemia and the development of cold and warm reactive erythrocyte-bound antibodies.
doi:10.1016/j.vetmic.2009.06.028
PMCID: PMC2771275  PMID: 19615832
Haemoplasma; Quantitative real-time PCR; Coombs’ test; Autoagglutination; Glucose
23.  Molecular Investigations of Rickettsia helvetica Infection in Dogs, Foxes, Humans, and Ixodes Ticks▿  
Applied and Environmental Microbiology  2009;75(10):3230-3237.
Rickettsia helvetica, a tick-borne member of the spotted-fever-group rickettsiae, is a suspected pathogen in humans; however, its role in animals is unknown. The aims of this study were to establish a R. helvetica-specific real-time TaqMan PCR assay and apply it to the analysis of tick vectors (to determine potential exposure risk) and blood samples from Canidae and humans (to determine prevalence of infection). The newly designed 23S rRNA gene assay for R. helvetica was more sensitive than a published citrate synthase gene (gltA) assay for several rickettsiae. Blood samples from 884 dogs, 58 foxes, and 214 human patients and 2,073 ticks (Ixodes spp.) collected from either vegetation or animals were analyzed. Although the maximal likelihood estimate of prevalence was 12% in unfed ticks and 36% in ticks collected from animals, none of the 1,156 blood samples tested PCR positive. Ticks from cats were more frequently PCR positive than ticks from dogs. Sequencing of the 23S rRNA and/or the gltA gene of 17 tick pools confirmed the presence of R. helvetica. Additionally, Rickettsia monacensis, which has not been previously found in Switzerland, was identified. In conclusion, R. helvetica was frequently detected in the tick population but not in blood samples. Nevertheless, due to the broad host range of Ixodes ticks and the high rate of infestation with this agent (i.e., R. helvetica was 13 times more frequent in unfed ticks than the tick-borne encephalitis virus), many mammals may be exposed to R. helvetica. The PCR assay described here represents an important tool for studying this topic.
doi:10.1128/AEM.00220-09
PMCID: PMC2681666  PMID: 19329665
24.  Feline Leukemia Virus and Other Pathogens as Important Threats to the Survival of the Critically Endangered Iberian Lynx (Lynx pardinus) 
PLoS ONE  2009;4(3):e4744.
Background
The Iberian lynx (Lynx pardinus) is considered the most endangered felid species in the world. In order to save this species, the Spanish authorities implemented a captive breeding program recruiting lynxes from the wild. In this context, a retrospective survey on prevalence of selected feline pathogens in free-ranging lynxes was initiated.
Methodology/ Principal Findings
We systematically analyzed the prevalence and importance of seven viral, one protozoan (Cytauxzoon felis), and several bacterial (e.g., hemotropic mycoplasma) infections in 77 of approximately 200 remaining free-ranging Iberian lynxes of the Doñana and Sierra Morena areas, in Southern Spain, between 2003 and 2007. With the exception of feline immunodeficiency virus (FIV), evidence of infection by all tested feline pathogens was found in Iberian lynxes. Fourteen lynxes were feline leukemia virus (FeLV) provirus-positive; eleven of these were antigenemic (FeLV p27 positive). All 14 animals tested negative for other viral infections. During a six-month period in 2007, six of the provirus-positive antigenemic lynxes died. Infection with FeLV but not with other infectious agents was associated with mortality (p<0.001). Sequencing of the FeLV surface glycoprotein gene revealed a common origin for ten of the eleven samples. The ten sequences were closely related to FeLV-A/61E, originally isolated from cats in the USA. Endogenous FeLV sequences were not detected.
Conclusions/Significance
It was concluded that the FeLV infection most likely originated from domestic cats invading the lynx's habitats. Data available regarding the time frame, co-infections, and outcome of FeLV-infections suggest that, in contrast to the domestic cat, the FeLV strain affecting the lynxes in 2007 is highly virulent to this species. Our data argue strongly for vaccination of lynxes and domestic cats in and around lynx's habitats in order to prevent further spread of the virus as well as reduction the domestic cat population if the lynx population is to be maintained.
doi:10.1371/journal.pone.0004744
PMCID: PMC2649436  PMID: 19270739
25.  RNase P RNA Gene (rnpB) Phylogeny of Hemoplasmas and Other Mycoplasma Species▿ † 
Journal of Clinical Microbiology  2008;46(5):1873-1877.
Partial sequences of the RNase P RNA gene (rnpB) were obtained from a number of hemoplasmas and other Mycoplasma species. Phylogenetic analysis of these sequences showed that all hemoplasmas were present within a single clade and were most closely related to Mycoplasma fastidiosum, similar to the results found with 16S rRNA gene phylogeny.
doi:10.1128/JCM.01859-07
PMCID: PMC2395117  PMID: 18337389

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