Inmaculada Amusategui

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.

Distribution and relationships between clinical and biopathological parameters in canine leishmaniasis.

Although the lesions, clinical signs and biochemical alterations observed in the course of canine leishmaniasis have been amply described a thorough definition and characterisation of the affected populations is important in order to detect relationships between parameters which may be involved in the development of this disease and to correctly assess further studies. This study included 61 dogs diagnosed with naturally acquired canine leishmaniasis by means of indirect immunofluorescence assay (IFA). At diagnosis, dogs were classified according to the following qualitative and quantitative variables: gender, breed, age, clinical picture, antibody titre, serum protein electrophoretogram, haemogram (CBC), urea, creatinine and ALT. Our population included dogs of 24 breeds, both sexes and different ages indicating no sex, age or breed predilection. In relation to the clinical picture, eight dogs were asymptomatic, 16 displayed mainly cutaneous signs, 18 presented primarily visceral signs and 19 displayed both cutaneous and visceral signs. Our results indicate that the clinical picture is significantly related to electrophoretogram and to RBC, PCV and haemoglobin. Dogs with mainly cutaneous signs showed the highest eosinophil mean values and those with mainly visceral signs showed the highest α-globulin mean values. This study confirms that the antibody titre is highly correlated with electrophoretogram and with RBC, PCV and haemoglobin. Lymphocytes were not associated or correlated with any other variable considered. PMNC, monocytes and eosinophils, as well as WBC, showed a significant correlation with β-globulins, which is difficult to interpret.

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.

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.

Seroprevalence of canine ehrlichiosis in Castilla-León (north-west Spain)

A study was undertaken of antibodies to Ehrlichia canis in dogs in the Comunidad Autonoma de Castilla-Leon (north-west Spain). A total of 308 serum samples, collected between January 1993 and May 1994, were analysed. The seroprevalence was 19.2 ± 2.24% (2 SE). The distribution of seropositive animals was not homogeneous in the different provinces; Salamanca had the highest seroprevalence (53.3 ± 25.2%). In relation to age, sex and evidence of tick infestation, no significant differences were noted. Dogs from rural areas (25.4 ± 8.1) presented higher prevalence than those from urban areas (17.3 ± 5.9) and dogs from kennels (9.5 ± 8.8). As far as the different functions of sampled dogs were concerned, guard and shepherd dogs had higher seroprevalence (28.5 ± 7.8%) than hunting (15.3 ± 7.6%) and companion (10.3 ± 6.1%) dogs.

Prospective comparative study of 3 treatment protocols using doxycycline or imidocarb dipropionate in dogs with naturally occurring ehrlichiosis.

This paper reports the clinicopathologic responses of 93 dogs with spontaneously occurring ehrlichiosis to 3 different treatment protocols. Thirty-two dogs were treated with doxycycline (10 mg/kg/day for 28 days), 31 were treated with imidocarb dipropionate (5 mg/kg given 15 days apart in 2 separate injections), and 30 were treated with both drugs simultaneously, at the doses as specified. The dogs underwent clinicopathologic evaluation before and after treatment, and were examined periodically during the 24-month period after the treatment. No differences were found in the clinical responses among the dogs in the 3 treatment groups. As for the clinicopathologic response, in spite of the fact that at the end of the study the results obtained with the 3 protocols were similar, the platelet count and serum protein electrophoresis results returned to normal more slowly in dogs that received imidocarb dipropionate as compared to those given the other 2 treatments.

Serological Evaluation of Anaplasma phagocytophilum Infection in Livestock in Northwestern Spain

Abstract:  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.

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.

Standardization of the diagnostic criteria for canine ehrlichiosis. Towards a universal case definition.

Abstract: Canine ehrlichiosis is a highly variable syndrome presenting a significant differential diagnostic difficulty. It imitates many metabolic and infectious diseases and lacks standardized diagnostic criteria, common reagents, and database resources. A clinical diagnosis of canine ehrlichiosis may be based on the manifestation of fever, thrombocytopenia, anorexia, nasolacrimal discharge, epistaxis, and exclusion of autoimmune and common canine vector borne diseases. These parameters are not invariably observed especially in the atypical form of the disease often caused by species other than Ehrlichia canis. A definitive diagnosis is based on the presence of specific antibodies to ehrlichial agent(s), the demonstration of the etiologic agent(s) itself, or specific amplicons by a strigently quality controlled PCR protocol. The relationship of the various clinical and laboratory parameters, the status of the currently available tests, and their real or presumed predictive value are discussed in the context of stimulating an effort to formulate an international standard for the diagnosis of this and related diseases of man and animals.