ObjectivesThe objectives of this study were to estimate the prevalence of feline haemoplasma infections in Northern Serbia, identify potential risk factors and perform molecular subtyping of feline immunodeficiency virus (FIV).MethodsPCR analysis for feline haemoplasmas was performed on surplus EDTA blood samples from 373 cats from the Belgrade region, Serbia. An ELISA was used to determine the prevalence of feline leukaemia virus (FeLV) and FIV; PCR was performed on a subpopulation of these cats. FIV subtyping was performed using PCR.ResultsWithin this population, 64/373 cats (17.2%) were infected with one or more haemoplasma species. Mycoplasma haemofelis was detected in 20/373 cats (5.4%), ‘Candidatus Mycoplasma haemominutum’ in 47/373 cats (12.6%) and ‘Candidatus Mycoplasma turicensis’ in 23/373 cats (6.2%). Coinfections were observed in 21/373 cats (5.6%). Based on ELISA serological retroviral testing, 4/310 cats (1.3%) were infected with FeLV, whereas 78/331 (23.6%) were infected with FIV. Multivariable analysis identified significant associations between haemoplasma infection and anaemia (anaemic/non-anaemic, odds ratio [OR] 2.7, 95% confidence interval [CI] 1.04–7.1; P = 0.041]), male gender (male/female, OR 4.5, 95% CI 2.22–9.03; P <0.0005), outdoor access (yes/no, OR 5.2, 95% CI 2.28–11.92; P <0.0005), non-pedigree breed (non-pedigree/pedigree, OR 5.5, 95% CI 1.24–24.84; P = 0.025) and FIV seropositive status (positive/negative, OR 2.4, 95% CI 1.21–4.83; P = 0.012). PCR analysis of the FIV ELISA-positive samples revealed clade D as being the most prevalent.Conclusions and relevanceAll three known species of feline haemoplasma were detected, confirming their presence in Serbia; ‘Candidatus Mycoplasma haemominutum’ was the most prevalent. We found a high prevalence of FIV-infected cats and FIV clade D was most prevalent.

Mycoplasma haemofelis (Mhf), ‘Candidatus Mycoplasma haemominutum’ (CMhm) and ‘Candidatus Mycoplasma turicensis’ (CMt) are agents of feline haemoplasmosis and can induce anaemia in cats. This study aimed to determine the prevalence and phylogeny of haemoplasma species in cats from Brazil's capital and surrounding areas, and whether correlation with haematological abnormalities existed. Feline haemoplasmas were found in 13.8% of 432 cats. CMhm was the most prevalent species (in 13.8% of cats), followed by Mhf (11.1%) and CMt (4.4%). Over 80% of haemoplasma-infected cats harboured two or more feline haemoplasma species: 7.1% of cats were co-infected with Mhf/CMhm, 0.4% with CMhm/CMt and 3.9% with Mhf/CMhm/CMt. Male gender was significantly associated with haemoplasma infections. No association was found between qPCR haemoplasma status and haematological variables, however CMhm relative copy numbers were correlated with red blood cell (RBC) numbers and packed cell volume (PCV). Haemoplasma 16S rRNA gene sequences (> 1 Kb) were derived from co-infected cats using novel haemoplasma species-specific primers. This allowed 16S rRNA gene sequences to be obtained despite the high level of co-infection, which precluded the use of universal 16S rRNA gene primers. Within each species, the Mhf, CMhm and CMt sequences showed > 99.8%, > 98.5% and > 98.8% identity, respectively. The Mhf, CMhm and CMt sequences showed > 99.2%, > 98.4% and > 97.8% identity, respectively, with GenBank sequences. Phylogenetic analysis showed all Mhf sequences to reside in a single clade, whereas the CMhm and CMt sequences each grouped into three distinct subclades. These phylogeny findings suggest the existence of different CMhm and CMt strains.

Autosomal-dominant polycystic kidney disease (ADPKD) is the most prevalent inherited genetic disease of cats, predominantly affecting Persian and Persian-related cats. A point mutation (C→A transversion) in exon 29 of the PKD1 gene causes ADPKD, and is the specific molecular target for genetic diagnosis in cats. The current study describes a newly developed touchdown polymerase chain reaction (PCR) to detect this single point mutation, using 2 primers specific for the mutant allele, adapted from an existing multiplex amplification refractory mutation system (ARMS PCR). Furthermore, correlations between the clinical outcomes of tested animals and the results of the genetic test were investigated. A total of 334 cats were tested, 188 from the Veterinary Hospital of Small Animals at the University of Brasilia, and 146 from an anti-rabies vaccine campaign of the Federal District. A total prevalence of 9% was evident among the samples, with 33% of the Persian cats testing positive, and 7% of the Brazilian long- and shorthaired cats testing positive. Prevalence was not correlated with gender or hemogram. Positive animals exhibited hyperglobulinemia ( P = 0.02). This research demonstrated that the mutation does not only occur in Persian and Persian-related cats, and that a touchdown PCR can be used to diagnose ADPKD.

Mycoplasma haemocanis (Mhc) and 'Candidatus Mycoplasma haematoparvum' (CMhp) are canine haemoplasma species that can induce anaemia in immunocompromised and/or splenectomised dogs. This study aimed to determine the prevalence and phylogeny of canine haemoplasma species in dogs from Nigeria and describe any risk factors for infection. Canine haemoplasma species-specific and generic haemoplasma qPCR assays were used. The species-specific qPCR assays found Mhc infection in 18 of 245 dogs (7.3%), and CMhp infection in only one dog (0.4%). The generic haemoplasma qPCR assays were positive in 44 of 245 (17.9%) dogs. Twenty-five dogs had discordant qPCR results in that they were generic haemoplasma qPCR positive but species-specific qPCR negative. Further evaluation of these dogs by 16S rDNA sequencing gave limited results but 5 were confirmed to be infected with non-haemoplasma species: 2 Anaplasma phagocytophilum, 1 Anaplasma ovis, 1 Serratia marcescens and 1 Aerococcus spp. The 16S rRNA gene sequences from Mhc species showed>99.8% identity with each other and>99.6% identity with GenBank sequences, and resided in a single clade with other global Mhc and Mycoplasma haemofelis sequences, indicating low 16S rRNA genetic variability amongst this canine haemoplasma species.

ABSTRACT.-Firmino F.P., Aquino L.C., Marçola T.G., Bittencourt M.V., Mycoplasma haemofelis is the agent of feline infectious anemia, although Candidatus M. haemominutum can also be associated. This study evaluated the frequency and hematological alterations caused by hemoplasma infections and co-infections with FeLV, FIV and Toxoplasma gondii in domestic cats from two distinct areas (urban -G1 and periurban -G2) of Brasília, Brazil. One hundred cats were evaluated, 51 from the G1 area and 49 from G2. No cats were positive for T. gondii. Hemoplasma infection was diagnosed in 33% cats from G1 and 32.6% from G2 (p>0.05). In G1 35.3% of the positive cats were infected with Mycoplasma haemofelis, 47.06% with Candidatus Mycoplasma haemominutum and 17.64% with mixed hemoplasma species infection; 12.5% of the cats identified as PCR positive in G2 were infected with Mycoplasma haemofelis, 18.75% with Candidatus Mycoplasma haemominutum and 68.75% with mixed infection. Cats from the periurban area had higher mixed hemoplasmas infection rates than those from urban area, and most of them were asymptomatic carriers. Cytology results were positive in only 5% of cats from G1. Mycoplasma haemofelis infected cats had normocytic normochromic anemia while the cats infected with Candidatus Mycoplasma haemominutum or with both species did not. 37.2% of G1 cats were co-infected with Mycoplasma haemofelis and FeLV, and presented lower PCV and hemoglobin concentration than those infected only with Mycoplasma haemofelis. The co--infection with Candidatus Mycoplasma haemominutum and FeLV produced lower WBC, segmented cells and platelets, and increased total protein concentration.