Assessment of Acute-Phase Protein Response Associated with the Different Pathological Forms of Bovine Paratuberculosis.

In this study, the concentrations of two acute-phase proteins (APPs), haptoglobin (Hp) and serum amyloid A (SAA), were quantitatively assessed in serum samples from cattle naturally infected with paratuberculosis (PTB). APP profiles were compared across 190 animals classified according to the different pathological forms associated with infection: uninfected (n = 59), with focal lesions (n = 73), multifocal lesions (n = 19), and diffuse paucibacillary (n = 11) and diffuse multibacillary lesions (n = 28). Our results showed a significant increase in both APPs in infected animals compared to the control group, with differences depending on the type of lesion. Hp and SAA levels were increased significantly in all infected animals, except in cows with diffuse multibacillary lesions that showed similar values to non-infected animals. The expression pattern of both APPs was similar and negatively correlated with the antibody levels against PTB. These results indicate that the release of Hp and SAA is related to the presence of PTB lesions associated with a high cell-mediated immune response and a lower bacterial load, suggesting that the pro-inflammatory cytokines that are associated with these forms are the main stimulus for their synthesis. These molecules could show some potential to be used as putative biomarkers of PTB infection, particularly for the identification of subclinical animals showing pathological forms related to latency or resistance to the development of advanced lesions.

Effects of Meloxicam on Hematologic and Plasma Biochemical Analyte Values and Results of Histologic Examination of Kidney Biopsy Specimens of African Grey Parrots (Psittacus erithacus).

In this study we evaluated the effects of meloxicam administered at 0.5 mg/kg IM q12h for 14 days on hematologic and plasma biochemical values and on kidney tissue in 11 healthy African grey parrots (Psittacus erithacus). Before treatment with meloxicam, blood samples were collected and renal biopsy samples were obtained from the cranial portion of the left kidney from each of the birds. On day 14 of treatment, a second blood sample and biopsy from the middle portion of the left kidney were obtained from each bird. All birds remained clinically normal throughout the study period. No significant differences were found between hematologic and plasma biochemical values before and after 14 days of treatment with meloxicam, except for a slight increase in median beta globulin and corresponding total globulin concentrations, and a slight decrease in median phosphorus concentration. Renal lesions were absent in 9 of 10 representative posttreatment biopsy samples. On the basis of these results, meloxicam administered at the dosage used in this study protocol does not appear to cause renal disease in African grey parrots.

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.

Evaluation of peripheral blood lymphocyte subsets in family-owned dogs naturally infected by Ehrlichia canis.

Previous research suggested that clinical manifestations, histopathological lesions, and even infection maintenance in the course of canine monocytic ehrlichiosis (CME) are directly related to the immune response developed by the host. In the present study, blood lymphocyte subsets were analyzed by multiparametric flow cytometry in 37 dogs with naturally occurring CME and 47 healthy dogs used as controls. T, T helper (Th), T cytotoxic (Tc), B, non-T, non-B lymphocytes and those that express MHC class II were characterized in every dog. Animals with CME showed higher relative values of T and Tc cells and a higher absolute number of Tc cells in peripheral blood. The percentage of Th cells and the absolute and relative values of B cells were higher in healthy animals than in CME-affected dogs. The significance of these changes on the pathogenesis of natural Ehrlichia canis infection in dogs needs further evaluation.

Immunophenotypic evaluation of working Labrador Retrievers and German Shepherd dogs living in the same environment.

Multiparametric flow cytometry was used to compare peripheral blood lymphocyte subset distribution between healthy working police Labrador Retrievers (LRs; n=12) and German Shepherd dogs (GS; n=11) living in the same environment. The CD4/CD8 ratio was significantly higher in LR than in GS because of the lower percentage of CD8+ T lymphocytes in LR. GS showed the highest relative percentage of CD3-/CD21- lymphocytes, whereas LR had the highest percentages of MHC II+ lymphocytes. Because age, sex, environmental and housing conditions, dietary patterns, and training or working routines were similar in both breeds in the study, differences in peripheral blood lymphocyte subset distribution could be attributed to the influence of breed on the immune system.

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 110 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 110. 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.

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.

Serological reactivity to Ehrlichia canis, Anaplasma phagocytophilum, Neorickettsia risticii, Borrelia burgdorferi and Rickettsia conorii in dogs from northwestern Spain.

The aim of the present work was to investigate the seroprevalence against Ehrlichia canis (Ec), Anaplasma phagocytophilum (Ap), Neorickettsia risticii (Nr), Rickettsia conorii (Rc), and Borrelia burgdorferi (Bb) in two different clusters of canine samples from Northwestern Spain. Cluster 1 included 479 dogs presented at veterinary clinics located in Ourense and Pontevedra. Cluster II included 170 dogs from the public kennel of Ourense. All 649 canine serum samples were analyzed by immunofluorescent antibody test. Prevalences against the above-mentioned agents in cluster I were: Rc (24.6%), Bb (6.26%), Ec (3.13%), Ap (5.01%), and Nr (1.04%), whereas for cluster II were: Rc (50%), Bb (8.8%), Ec (54.7%), Ap (45.3%), and Nr (4.7%). Rc was significantly associated with age and history of exposure to ticks, and Bb showed a statistical relationship with age and clinical status. Ec and Ap were related to the occupation of the dogs, with stray dogs being the most frequently seropositive. Furthermore, seroreactivity against Ec and Ap was significantly higher in Ourense than in Pontevedra. The univariate analysis demonstrated a significant concomitant seroreactivity between Ec and Ap and between Rc and Ec and Ap antigens. The seroreactivity to Nr must be interpreted very cautiously as this infectious agent has been seldom reported outside North America.

Immunophenotype of dogs with subclinical ehrlichiosis.

The purpose of this study was the characterization of the different subsets of lymphocyte cells in dogs with subclinical ehrlichiosis in order to contribute to the knowledge of the immune response developed in the course of this disease. Thirty-eight dogs with subclinical ehrlichiosis and 15 unaffected dogs from two shelters in the area of Valencia (eastern Spain) were included in the study. The study of lymphocyte populations was made by flow cytometry. Monoclonal antibodies against CD3, CD4, CD8, and CD21 were used. Based on our results, the most common findings of the subclinical phase of canine ehrlichiosis were lymphocytosis, relative neutropenia, and a decrease in the neutrophil/lymphocyte ratio. Lymphocytosis in these dogs was from an increase of T lymphocyte counts. Tc cell counts in dogs with subclinical ehrlichiosis were higher than in healthy dogs. This rise in the number of Tc lymphocytes resulted in a reduced percentage of Th lymphocytes and in a decrease in the CD4/CD8 ratio.

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.

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.

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.

Serological evaluation of Anaplasma phagocytophilum infection in livestock in northwestern Spain.

A total of 1,098 serum samples were analyzed against Anaplasma phagocytophilum by immunofluorescent antibody (IFA) test. These serum samples belonged to four different populations distributed throughout two provinces of Galicia (Ourense and Pontevedra) located in northwestern Spain: bovine population (456 samples); ovine population (389 samples); caprine population (207 samples); and equine population (46 serum samples, all from Pontevedra). The seroprevalence against A. phagocytophilum within the bovine population was 3.07%. On the other hand, two of 389 (0.51%) sheep and one of 207 (0.48%) goats tested were seropositive, all of them showing low antibody titer. Seroprevalence within the equine population was 6.52% (3/46). Our results reveal the presence of antibodies against A. phagocytophilum in livestock from northwestern Spain, mainly in Pontevedra.

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 Pallas's 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.

Assessment of feline ehrlichiosis in central Spain using serology and a polymerase chain reaction technique.

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.

Seroprevalence of Leishmania infantum in northwestern Spain, an area traditionally considered free of leishmaniasis.

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.

First isolation and molecular characterization of Ehrlichia canis in Spain.

This paper reports the first isolation and culture of Ehrlichia canis in Spain from a naturally infected dog using the DH82 cell line. After DNA extraction and PCR amplification, a nearly complete (1412bp) sequence of the 16S rRNA gene of the new E. canis strain was obtained. The GenBank accession number for the nucleotide sequence of this strain is AY394465. This sequence was aligned with the 16S rRNA gene sequences of other Ehrlichia strains accessible in GenBank. The 16S rRNA gene sequence of the E. canis strain reported here showed a high percentage of similarity with the 16S rRNA gene sequence of E. canis from different geographic areas including Japan, Venezuela and Israel. These data confirm the presence of E. canis in Spain.