Enara Aguirre

First report of an intraerythrocytic small piroplasm in wild Iberian lynx (Lynx pardinus).

A wild injured Iberian lynx (Lynx pardinus) was taken from the Sierra Morena population. During the health check small intraerythrocytic piroplasms, morphologically indistinguishable from other feline piroplasms, were observed in Wright-Giemsa-stained blood films. Amplification by polymerase chain reaction of a portion of the 18S nuclear small subunit (NSS) rRNA gene and sequencing revealed similarity of the unknown organism with sequences obtained from Pallass cat from Mongolia and from a domestic cat in Spain. In a retrospective (1993–2003) study of 50 Iberian lynx tissue samples, no amplifications of the 18S NSS rRNA gene of the organism were obtained. This is the first report of a naturally occurring erythroparasitemia in the Iberian lynx and the first documented case of naturally occurring piroplasm infection in a free-ranging felid from Europe.

Seroprevalence of Leishmania infantum in Northwestern Spain, an Area Traditionally Considered Free of Leishmaniasis

Abstract: Northwestern Spain has traditionally been considered to be free from leishmaniasis. The aim of this work was to determine the prevalence of canine leishmaniasis in this area and to assess the influence of several risk factors on the incidence of this disease. A total of 479 dogs attended at different veterinary clinics in northwestern Spain were tested for L. infantum with the immunofluorescent antibody (IFA) test. The seroprevalence of L. infantum in this area was 3.7%. Most of the seropositive dogs lived in two locations: Valdcorras (seroprevalence of 29.2%) and Ourense (seroprevalence of 7.5%). The detection of high antibody titers in most of the seropositive dogs (many of which presented clinical signs) coupled with the certainity that some of these dogs had never been outside their home areas indicates the presence of this zoonosis in these two sites. On the other hand, companion dogs were significantly less likely to acquire the disease than sheep dogs, hunting dogs, and those from kennels.

Assessment of Feline Ehrlichiosis in Central Spain Using Serology and a Polymerase Chain Reaction Technique

Abstract: Antibodies to Ehrlichia spp. and inclusion bodies compatible with Ehrlichia spp. in feline blood cells have been previously detected in Spain. The aim of this study was to assess the presence of antibodies to E. canis, N. risticii, and A. phagocytophilum in 122 feline serum samples from Madrid (central Spain). In addition, Ehrlichia genus‐specific, one‐tube, nested polymerase chain reaction (PCR) was performed from blood samples from these cats. Of the cats, 10.6% were seropositive for E. canis, 2.4% were positive for N. risticii, and 4.9% were seropositive for A. phagocytophilum. Two N. risticii‐positive cats and one animal seropositive to A. phagocytophilum were also seropositive for E. canis. Despite these seropositive results, all the blood samples analyzed by PCR were negative. Our results demonstrate reactivity against agents implicated in feline ehrlichiosis in Spain. Further studies should be performed in order to clarify the significance of serology and PCR in the diagnosis of feline ehrlichiosis.

Development of a sensitive and specific indirect enzyme-linked immunosorbent assay based on a baculovirus recombinant antigen for detection of specific antibodies against Ehrlichia canis.

An indirect enzyme-linked immunosorbent assay (ELISA) based on baculovirus recombinant P30 protein of Ehrlichia canis and the 1BH4 anticanine IgG monoclonal antibody was developed and evaluated by examining a panel of 98 positive and 157 negative sera using the indirect fluorescent antibody (IFA) test as the reference technique. The P30-based ELISA appeared to be sensitive and specific (77.55% and 95.54%, respectively) when qualitative results (positive/negative) were compared with those of the IFA test; the coefficient of correlation (R) between the 2 tests was 0.833. Furthermore, it was possible to establish a mathematical formula for use in comparing the results of both techniques. These results indicate that recombinant P30 antigen-based ELISA is a suitable alternative of the IFA test for simple, consistent, and rapid serodiagnosis of canine ehrlichiosis. Moreover, the use of this recombinant protein as antigen offers a great advantage for antigen preparation in comparison with other techniques in which the whole E. canis organism is used as antigen.

Comparison between Different Polymerase Chain Reaction Methods for the Diagnosis of Ehrlichia canis Infection

The aim of this study was to compare different polymerase chain reaction (PCR) methods for the detection of Ehrlichia canis in blood samples and to relate these results to clinical findings and serology to E. canis using the indirect fluorescent antibody (IFA) test. Nine seropositive and nine seronegative dogs were included in this study. DNA was extracted once and used in one simple PCR and five nested PCR protocols previously described. In selected dogs (three seropositive and one seronegative) blood samples were aseptically collected in order to attempt the isolation of E. canis in the DH82 cell line. Results show that nested PCR protocols seem to be more sensitive than the simple PCR. Considering only nested PCR protocols, 33% of the IFA‐positive samples were PCR positive using the five different protocols. The rest of the IFA‐positive samples were PCR positive or negative depending on the protocol used. Clinical signs and laboratory findings compatible with canine monocytic ehrlichiosis (CME) were found in 67% of dogs positive by the IFA test. All samples positive by both techniques (IFA test and PCR) were from dogs suffering from clinical CME. IFA‐negative samples were PCR negative, except 22% that were PCR positive when using only one of the nested PCR protocols. Isolation of the agent was exclusively achieved in the only case in which the IFA test and all the PCR protocols were also positive.

Results from an indirect fluorescent antibody test using three different strains of Ehrlichia canis

An indirect fluorescent antibody (IFA) test is usually performed to detect antibodies in dogs naturally infected by Ehrlichia canis. In this work, results obtained using three different E. canis strains as antigen (a commercial antigen, the E. canis Oklahoma strain and the E. canis Madrid strain) were compared. One hundred and forty-nine serum samples obtained from dogs living in the centre of Spain were analysed. When qualitative results were evaluated, identical results were detected in 87.2% of samples for the three antigens tested. When comparing antibody titre results, differences between the Madrid strain and the commercial antigen, and between the Madrid and Oklahoma strains were statistically significant (P<0.0001). No differences were found when comparing the Oklahoma strain with the commercial antigen (P=0.562). Subtle intra-laboratory variations shown in this study suggest a higher sensitivity of the IFA test when an autochthonous strain is used as antigen.