Bartonella chomelii is the most frequent species infecting cattle grazing in communal mountain pastures in Spain.

The presence of Bartonella spp. was investigated in domestic ungulates grazing in communal pastures from a mountain area in northern Spain, where 18.3% (17/93) of cattle were found to be positive by PCR combined with a reverse line blot (PCR/RLB), whereas sheep (n = 133) or horses (n = 91) were found not to be infected by this pathogen. Bartonella infection was significantly associated with age, since older animals showed a higher prevalence than heifers and calves. In contrast to other studies, B. chomelii was the most frequent species found in cattle (14/17), while B. bovis was detected in only three animals. Moreover, 18 B. chomelii isolates and one B. bovis isolate were obtained from nine animals. Afterwards, B. chomelii isolates were characterized by a multilocus sequence typing (MLST) method which was adapted in this study. This method presented a high discrimination power, identifying nine different sequence types (STs). This characterization also showed the presence of different STs simultaneously in the same host and that STs had switched over time in one of the animals. In addition, B. chomelii STs seem to group phylogenetically in two different lineages. The only B. bovis isolate was characterized with a previously described MLST method. This isolate corresponded to a new ST which is located in lineage I, where the B. bovis strains infecting Bos taurus subsp. taurus are grouped. Further studies on the dynamics of Bartonella infection in cattle and the potential ectoparasites involved in the transmission of this microorganism should be performed, improving knowledge about the interaction of Bartonella spp. and domestic ungulates.

Variability of Bartonella genotypes among small mammals in Spain.

In order to study which Bartonella genotypes are circulating among small mammals in Spain, we analyzed the spleens of 395 animals from three different areas-247 animals from the Basque Country (northern Spain), 121 animals from Catalonia (northeastern Spain), and 27 animals from Madrid (central Spain)-by a triplex PCR combined with a reverse line blot previously described by our group. The prevalence of Bartonella was 26.8% (106/395), and in 4.8% (19/395) of the animals more than one Bartonella genotype was detected. The study of gltA and the intergenic transcribed spacer in the positive samples demonstrated a large diversity, allowing the assignation of them into 22 genotypes. The most prevalent genotypes were 2 and 3, which are closely related to Bartonella taylorii. In addition, nine genotypes were associated with specific mammal species. Genotypes close to the zoonotic Bartonella grahamii, Bartonella elizabethae, and Bartonella rochalimae were also detected. Ten genotypes showed a percentage of similarity with known Bartonella species lower than 96%, suggesting the presence of potential new species. Further studies of the impact of these pathogens on human health and especially in cases of febrile illness in Spain are strongly recommended. Furthermore, our method has been updated with 21 new probes in a final panel of 36, which represents a robust molecular tool for clinical and environmental Bartonella studies.

A new chemical preservative that permits analysis of urine sediment for light microscopic examination 12 h after emission.

INTRODUCTION: Urine diagnosis by light microscopy is considerably more difficult when specimens are analyzed after a certain period of time. OBJECTIVES: (1) To investigate whether this change effectively exists at a significant level of 12 h. (2) To apply measures, once the above has been done, allowing for the analysis of samples beyond 12 h in similar conditions. MATERIALS AND METHODS: Both freshly produced urine and pathological samples were used under certain experimental conditions: initial, 12 h, 12 h + fridge at 4 degrees C (F), 12 h + chemical preservation (S) and 12 h + SF. The chemical preservative was prepared at a 50/50 ratio with 3% formaldehyde and 2.5% glutaraldehyde by the addition of a buffer of pH 7.2-7.4, resulting in a solution of pH 7.35 at 25 degrees C room temperature. Urinalysis was carried out on all samples: glucose (enzymatic method of hexokinase) and total protein in liquid (red pyrogallol method). Centrifugation was followed by sediment analysis with light microscopy. Statistical analysis was done with the Kolmogorov-Smirnov normality test, Friedman nonparametric test and multiple comparisons. RESULTS AND CONCLUSION: Urine samples tested 12 h after having been produced changed significantly (p<0.0001), making it necessary to adopt certain measures to maintain their initial conditions. In our case, after the addition of the chemical preservative, samples did not present any changes (p>0.10) in relation to the initial conditions and were seen to be reliable, therefore indicating the suitability and effectiveness of the analytical conditions (urinalysis in particular, sediment analysis).