Fernando Rodríguez-Franco

Serologic and Molecular Evaluation of Leishmania infantum in Cats from Central Spain

Infection by different Leishmania spp. in cats has been reported in many countries. In Spain, since the first Leishmania infection described in 1933, sporadic clinical cases in cats have been reported. Various serologic studies performed in other areas of Spain have shown seroprevalences ranging between 1.7 and 60%. The aim of the present study was to determine the prevalence of leishmaniasis in cats from Central Spain (Madrid), and to assess the existence of associations between Leishmania infantum infection and relevant data obtained from each cat. Two‐hundred thirty‐three cats attended at the Veterinary Teaching Hospital in Madrid between September 2005 and June 2006 were tested for L. infantum using the indirect immunofluorescent antibody (IFA) test (cutoff: 1:100) and PCR. PCR testing was performed on the samples to detect Leishmania infection, targeting the kinetoplast DNA (kDNA). Our results showed a seroprevalence of 1.29% (3/233) using IFA test. Another seven cats were also seroreactive to L. infantum one dilution under the cutoff (1:50). Considering all the seroreactive samples, the percentage of positive animals to L. infantum was 4.29%. Only one of the cats (0.43%) included in the study was PCR‐positive. Relative lymphocytosis and an increase in alanine aminotransferase (ALT) value were statistically associated with seroreactivity to L. infantum. Our results demonstrate the presence of cats seroreactive to L. infantum in Central Spain, an endemic area for this disease in dogs.

Vector-Borne Diseases in Client-Owned and Stray Cats from Madrid, Spain

The role of various vector-borne pathogens as a cause of disease in cats has not been clearly determined. The current study evaluated risk factors, clinical and laboratory abnormalities associated with Ehrlichia spp., Anaplasma spp., Neorickettsia spp., Leishmania spp., and Bartonella spp. infection or exposure in 680 client-owned and stray cats from Madrid, Spain. Our results indicate that a large portion (35.1%) of the cat population of Madrid, Spain, is exposed to at least one of the five vector-borne pathogens tested. We found seroreactivity to Bartonella henselae in 23.8%, to Ehrlichia canis in 9.9%, to Anaplasma phagocytophilum in 8.4%, to Leishmania infantum in 3.7%, and to Neorickettsia risticii in 1% of the feline study population. About 9.9% of cats had antibody reactivity to more than one agent. L. infantum DNA was amplified from four cats (0.6%), B. henselae DNA from one cat (0.15%), and B. clarridgeiae DNA from another cat (0.15%).

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.

Detection of perinuclear antineutrophil cytoplasmic antibodies and antinuclear antibodies in the diagnosis of canine inflammatory bowel disease.

In recent years, serologic markers for diagnosis and classification of inflammatory bowel disease (IBD) have been used in human medicine. Perinuclear, antineutrophil, cytoplasmic antibodies (p-ANCA) are the most important of these markers. Because of their similar pattern of fluorescence, antinuclear antibodies (ANA) could cause misleading interpretations. The aim of the present study was to evaluate the use of an indirect fluorescent antibody test to detect p-ANCA in dogs with IBD, to compare the presence of p-ANCA in dogs with IBD with the presence of the same antibodies in other dogs, and to analyze the presence of ANAs in the p-ANCA-positive samples. Using a 1:10 dilution as a cutoff point, a sensitivity of 0.34 and a specificity of 0.86 was obtained when dogs with IBD were compared with the other groups as a whole, and specificity increased to 0.94 when dogs with IBD were compared with animals with other chronic gastrointestinal disorders. The lowest specificity value, 0.76, was obtained when the group of dogs with IBD was compared with that of dogs with different inflammatory and infectious disorders. Globally, 78 dogs were positive for p-ANCA when the cutoff was 1:10. Only 1 dog from these 78 animals was also seropositive to ANA. The results suggest that 1) detection of p-ANCA might be included in the IBD diagnostic protocol as another test to differentiate between this disease and other digestive diseases with similar clinical signs, and 2) most p-ANCA- positive dogs are not ANA positive.

Evaluation of perinuclear antineutrophilic cytoplasmic antibodies in sera from dogs with inflammatory bowel disease or intestinal lymphoma

OBJECTIVE To assess and compare the expression of perinuclear antineutrophilic cytoplasmic antibodies (pANCA) in sera obtained from dogs with inflammatory bowel disease (IBD) and dogs with intestinal lymphoma. ANIMALS 104 dogs with IBD and 23 dogs with intestinal lymphoma. PROCEDURES Each ill dog had persistent gastrointestinal signs (> 3 weeks in duration) and absence of response to diet changes or antimicrobial treatments. Gastrointestinal endoscopy was performed in ill dogs to obtain intestinal biopsy specimens for histologic confirmation of IBD or lymphoma. A serum sample was obtained from each ill dog. Neutrophils were isolated from a blood sample from the healthy dog; neutrophil-bearing slides were incubated with serum from each ill dog and examined for expression of pANCA by use of an indirect immunofluorescence technique. Detection of cells that had a perinuclear fluorescence pattern was considered a positive result. RESULTS The 2 groups of dogs did not differ with regard to breed and sex but did differ with regard to age. Expression of pANCA was detected in 38 of the 104 (36.5%) dogs with IBD and 4 of the 23 (17.4%) dogs with intestinal lymphoma. Although the frequency of pANCA expression was higher in dogs with IBD, compared with findings in dogs with intestinal lymphoma, the difference was not significant. CONCLUSIONS AND CLINICAL RELEVANCE Results indicated that circulating pANCA are present in some dogs with IBD or intestinal lymphoma. However, pANCA detection does not seem to be useful for distinguishing dogs with IBD from dogs with intestinal lymphoma.

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.

White spots on the mucosal surface of the duodenum in dogs with lymphocytic plasmacytic enteritis.

Distended lacteals, described as expanded white villi in duodenum, are strongly indicative of primary intestinal lymphangiectasia. In the present study, we evaluated the significance of white spots present in the duodenal mucosa of dogs with lymphocytic plasmacytic enteritis (LPE). Fifty dogs with LPE were included in this study, and white spots were detected in the duodenal mucosa in 22 dogs during endoscopy. Hypoproteinemia was more frequent in dogs with white spots than in dogs without spots (p = 0.02). Serum protein and albumin concentration were significantly lower in LPE dogs with white spots (p = 0.038) compared to LPE dogs without white spots (p = 0.039). There was a significant correlation between white spots density and lymphatic dilatation histological scores (p = 0.023; ρ = 0.481). These results suggest that the presence of white spots in the duodenal mucosa of dogs is not a finding exclusive for intestinal lymphangiectasia. Low serum protein and albumin concentrations together with lymphatic dilatation seem to be related to the presence of white spots in the duodenal mucosa of LPE dogs.

Effects of doxycycline on haematology, blood chemistry and peripheral blood lymphocyte subsets of healthy dogs and dogs naturally infected with Ehrlichia canis.

Canine monocytic ehrlichiosis (CME), caused by Ehrlichia canis, is a vector-borne disease with a worldwide distribution. It has been proposed that the pathogenesis, clinical severity and outcome of disease caused by Ehrlichia spp. can be attributed to the immune response rather than to any direct rickettsial effect. Moreover, doxycycline, the antimicrobial of choice for the treatment of CME, has immunomodulatory and anti-inflammatory properties associated with blood leukocyte proliferation function, cytokine synthesis, and matrix metalloproteinase activity. In order to assess the potential effects of doxycycline, dependent and independent of its antimicrobial activity, the present study compared changes in haematology, blood chemistry and circulating lymphocyte subpopulations in 12 healthy dogs and 20 dogs with CME after doxycycline therapy. Some changes were recorded only in the CME affected dogs, probably due to the antimicrobial effect of doxycycline. However, increases in mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, platelet count and α2-globulins, and decreased plasma creatinine were observed in both healthy and CME affected dogs. The absolute count of B lymphocytes (CD21(+)) increased initially, but then decreased until the end of the study period in both groups. A potential effect of doxycycline unrelated to its antimicrobial activity against E. canis is suggested, taking into account the results observed both in healthy dogs and in dogs with CME.

Early-life longitudinal survey of peripheral blood lymphocyte subsets in Beagle dogs.

A longitudinal study of peripheral blood lymphocyte subsets from 7 to 15 months of age was performed in Beagle dogs employing a multiparametric flow cytometry. The data were compared with data obtained from adult Beagle dogs that were housed in the same animal facilities and that were subjected to the same controls during the 34 weeks of the study. Absolute counts of total lymphocytes and CD3+ T, CD3+CD4+ Th and CD21+ B lymphocytes decreased during the entire 34 weeks period of the study in the young dogs group. The same was observed with regard to the percentage of CD3+CD4+ Th lymphocytes and the CD4/CD8 ratio, while the percentage of CD3+CD8+ Tc lymphocytes increased from 7 to 15 months of age. These age-related changes found in lymphocyte subsets distribution of young dogs led to level the absolute and relative values of adult dog lymphocytes. The observations of this longitudinal study illustrate the changes related to maturation of lymphocyte subsets that occur during early life in Beagle dogs.

Evaluation of lymphocyte populations in dogs naturally infected by Ehrlichia canis with and without clinical signs

Immune response elicited by the host during ehrlichial infections could influence the clinical signs and laboratory and pathological findings. Twenty-eight dogs naturally infected by Ehrlichia canis were included in this study. Twenty of them presented only laboratory findings traditionally associated with canine monocytic ehrlichiosis (CME), whilst 8 dogs also showed clinical signs classically associated with CME (pale mucous membranes, fever, lymphadenopathy, weight loss, anorexia, lethargy or signs attributable to bleeding tendencies). A multiparametric flow cytometric study was performed to analyze the distribution of the main lymphocyte subsets (T, Th, Tc, B, and those that express MHC class II) in the peripheral blood. Statistically significant differences between dogs naturally infected by E. canis in a clinical or subclinical stage were not detected when evaluating lymphocyte subsets in peripheral blood samples. Dogs with clinical signs showed lower relative and absolute values of B lymphocytes than dogs without clinical signs, although the differences were not statistically significant.