Haemoplasma copy quantity quantification was derived by comparison to a standard curve generated by amplification of plasmids containing cloned 16S rDNA PCR products from each of the haemoplasma varieties (Tasker et al
Haemoplasma copy quantity quantification was derived by comparison to a standard curve generated by amplification of plasmids containing cloned 16S rDNA PCR products from each of the haemoplasma varieties (Tasker et al., 2003; Willi et al., 2005; Peters et al., 2008). 2.4. M. haemominutum and M. turicensis (Foley and Pedersen, 2001; Neimark et al., 2001, 2002; Willi et al., 2005, 2006). illness often causes haemolysis (Berent et al., 1998; Foley et al., 1998; Westfall et al., 2001) but M. haemominutum illness does not usually result in clinically significant anaemia (Foley et al., 1998; Westfall et al., 2001; Tasker et al., 2006a) without concurrent immunocompromisation (George et al., 2002). Experimental M. turicensis offers only been evaluated in one earlier study in which two pet cats developed anaemia secondary to illness (Willi et al., 2005) with more severe anaemia developing in the one cat that had been immunocompromised before inoculation. SA-2 Positive Coombs (direct antiglobulin) checks, indicating the presence of erythrocyte-bound antibodies, have been reported with haemoplasma infections in a number of varieties, including pet cats (Bundza et al., 1976; Harvey and Gaskin, 1978; Hoffmann et al., 1981). It has been proposed that such antibodies may contribute to the haemoplasma-associated haemolysis. However, investigations describing Coombs checks in haemoplasma-infected pet cats possess assorted greatly with respect to the methods explained, or used, to perform Coombs screening (reagents, dilutions, temps used and timing of sample collection) (Maede et al., 1974; Maede and Hata, 1975; Zulty and Kociba, 1990; Alleman et al., 1999; Willi et al., 2005). Additionally, studies have not yet described Coombs results in pet cats infected with each of the three feline haemoplasma varieties. Severe hypoglycaemia has also been reported in several animal varieties acutely infected with haemoplasmas; sheep (M. haemodidelphidis) (Messick et al., 2000), pigs (M. haemolamae) (McLaughlin et al., 1990; Messick et al., 2002) and calves (M. Salvianolic acid F haemominutum or M. turicensis. 2.?Materials and methods 2.1. Study design and protocol Sixteen barrier-maintained specific pathogen (retroviral) free-derived domestic-shorthaired pet cats, aged 7 weeks, were used. Due to the Salvianolic acid F variance in haemoplasma copy number anticipated with illness (Tasker et al., 2003, 2006b), Salvianolic acid F and the undertaking of a parallel study investigating subsequent haemoplasma copy quantity variance in the blood and cells of some of the same pet cats (Tasker et al., in preparation), more pet cats were infected with than the additional two varieties. Ten pet cats were randomly assigned to the group; Group HF (pet Salvianolic acid F cats HF1 to HF4, HF6 to HF10 and HF12; six entire females and four neutered males), three to the M. haemominutum group; Group HM (pet cats HM1, HM2 and HM4; one entire woman and two neutered males), and three to the M. turicensis group; Group TU (pet cats TU1, TU2 and TU4; three neutered males). The discontinuous numbering of the pet cats was due to the inclusion of additional pet cats in the parallel study (Tasker et al., in preparation) mentioned above. Blood samples were collected on Day time 0 of the study, immediately before haemoplasma inoculation. Blood was placed into EDTA-anticoagulant for total blood count using a Cell Dyn 3700 analyser (Abbott, IL, USA), packed cell volume (PCV) dedication (microhaematocrit tube centrifugation) and haemoplasma real-time quantitative PCR (qPCR), whilst a drop of remaining non-anticoagulated blood was utilized for blood glucose measurement (Accu-chek Aviva blood glucose system, Roche Diagnostics Ltd., Lewes, East Sussex, UK). Experimental inoculation of all pet cats with the respective haemoplasma varieties was carried out using heparinised blood collected from three barrier-maintained carrier donor pet cats. Cat blood types were predetermined (RapidVet-H blood typing cards, DMS Laboratories Inc., New Jersey, US) to be compatible (all type A). Following collection, the heparinised blood was placed on damp snow and 2?ml of heparinised donor blood was given intravenously to each of the 16 pet cats, via pre-placed cephalic intravenous catheters, within 5?min of collection. The haemoplasma inoculum doses comprised 1.55??108 copies, 5.04??106 M. haemominutum copies or 2.42??106 M. turicensis copies per cat. Blood samples were collected from all pet cats three times weekly from 2 to 85 days post-infection (DPI): twice weekly, 0.3?ml blood was utilized for PCV, haemoplasma qPCR and blood glucose measurement, and once weekly, 1?ml blood was utilized for haematological exam, Coombs screening, PCV, haemoplasma qPCR and blood glucose measurement..