L. Del Rio

Field evaluation of a new commercially available ELISA based on a recombinant antigen for diagnosing Chlamydophila abortus (Chlamydia psittaci serotype 1) infection.

A new commercially available ELISA (ELISAr-Chlamydia) for detecting antibodies against Chlamydophila abortus has been evaluated using sheep field serum samples. The ELISA is based on a recombinant antigen which expresses part of a protein from the 80-90kDa family that is specific to C. abortus. Sera (105) from six flocks with confirmed ovine chlamydial abortion (OEA) outbreaks were used in this study, as well as sera (258) from 18 flocks which had suffered no OEA in the last lambing. The ELISAr-Chlamydia was compared with the complement fixation test (CFT) and with an ELISA using purified C. abortus elementary bodies (ELISA-EB), employing as reference technique a comparative microimmunofluorescence test that differentiates C. abortus infection from Chlamydophila pecorum infection. The results showed that the sensitivity of ELISAr-Chlamydia was 90.9% with a specificity of 85.9%, the sensitivity of CFT was 71.0% with a specificity of 83.6%, while the sensitivity of ELISA-EB was 95.2% and the specificity was 54.2%. Furthermore, ELISAr-Chlamydia was the test with fewer false positives resulting from positive reactivity to C. pecorum, although 15% of the sera positive for C. pecorum but negative for C. abortus antibodies reacted positively. This study demonstrated with field material that ELISAr-Chlamydia provides the most balanced results between sensitivity and specificity, especially in flocks with no clinical OEA but reactivity to C. abortus.

Cryptic Leishmaniosis by Leishmania infantum, a feature of canines only? A study of natural infection in wild rabbits, humans and dogs in southeastern Spain

An epidemiological study was carried out to investigate asymptomatic Leishmania infantum infection by PCR and ELISA in wild rabbits, humans and domestic dogs in southeastern Spain. Seroprevalence was 0% (0/36) in rabbits, 2% (13/657) in humans and 7% (14/208) in dogs. The prevalence of PCR-positives was 0.6% (1/162) in rabbits tested in a wide range of tissue samples, 2% (8/392) in humans analysed in blood samples and 10% (20/193) and 67% (29/43) in dogs analysed in blood and lymphoid tissue samples, respectively. Results suggest that wild rabbits have a very low risk of becoming chronically infected with L. infantum, and provide further evidence that cryptic L. infantum infection is widespread in the domestic dog population and is also present in a comparatively smaller proportion of healthy humans. The epidemiological and clinical implications of these findings are discussed.

Questionnaire survey on Canine Leishmaniosis in southeastern Spain

A veterinary practitioners questionnaire survey on Canine Leishmaniosis (CanL) was carried out in southeast Spain to provide information on disease incidence, diagnosis and recommended preventive measures. A third of 101 responding veterinarians reported seeing more than 20 CanL cases during the previous year and clinical signs considered frequent or very frequent included weight loss, areas of exfoliative dermatitis, lymphadenomegaly, kidney dysfunction, onychogryphosis, cutaneous ulcers, anaemia, asthenia and apathy. Almost a third of veterinarians reported diagnosing CanL solely on the basis of lack of response to treatment for other chronic illnesses. The frequency of individual signs did not always reflect their diagnostic value. The majority of veterinarians, considered lymphadenomegaly, exfoliative dermatitis, onychogryphosis, weight loss and epistaxis to be the best disease indicators. However, up to 93% of veterinarians reported using laboratory tests to confirm infection, including indirect (biochemical and antibody assays) and direct (PCR and microscopy) methods. Among recommended measures to prevent CanL, insecticide impregnated collars and topical spot-on capsules were prescribed by 92% and 74% of respondents; however, at least 11% of veterinarians believed existing CanL preventive measures were ineffective. Multivariable statistical analysis indicated that seeing more than 20 CanL cases during the previous year was independently associated with being located in Alicante province, having a rural clientele and recognising that dogs with no other sign but a lack of response to other chronic disease treatments may be infected with CanL. There was a weak association between CanL incidence and rainfall in the months prior to typical sandfly peak abundance.

Evaluating the efficacy of cinnamaldehyde and Echinacea purpurea plant extract in broilers against Eimeria acervulina.

Coccidiostats could be phased out as feed additives before 1 January 2013 for public health and food safety reasons, and, as a replacement, bioactive compounds found in plants are currently being investigated since they are more likely to be found acceptable by consumers. The aim of our study was to evaluate the effect of cinnamaldehyde (CIN) and Echinacea purpurea plant extract (EP) as additives by analyzing the performance traits, oocyst excretion and intestinal lesions following experimental infection with Eimeria acervulina. A total of 72 Ross male broilers were raised from 1 to 35 d and randomly assigned to four dietary treatments: control, without additives (C); 150 mg kg(-1) cinnamaldehyde (CIN); 1000 mg kg(-1)E. purpurea plant extract (EP); 150 mg kg(-1) cinnamaldehyde plus 1000 mg kg(-1)E. purpurea plant extract (CIN+EP). At 25 d, 12 chickens per treatment were orally infected with E. acervulina. Coccidia infestation led to lower performance but with no significant differences between the infected groups. Oocyst output reached its peak from 6 to 9 d post-infection in all treatments. At duodenal level, gross lesion scores were lower for cinnamaldehyde diets (P<0.05). A similar trend was observed in the microscopic lesion scores, with a non-significant reduction as a result of cinnamaldehyde addition (P>0.05). Scoring methods for macro- and microscopic lesions showed a positive linear relationship (G=+0.70). Further studies are necessary to assess the possible anticoccidian action of the cinnamaldehyde and its value as an alternative or adjunct in therapeutic or prophylactic strategies.

Evidence for widespread Leishmania infantum infection among wild carnivores in L. infantum periendemic northern Spain

Leishmania spp. infection was investigated in tissue samples of wild carnivores from the Spanish Basque Country (BC), by PCR and DNA sequencing. The region is at the northern periphery of Leishmania infantum endemic Iberian Peninsula and infection in the dog (reservoir) or other species has not been previously reported. Leishmania kinetoplast DNA was detected by real-time PCR (rtPCR) in 28% (44/156) of animals. Specifically, in 26% of Eurasian badgers (n=53), 29% of foxes (n=48), 29% of stone martens (n=21) and in 25-50% of less numerous species including genets, wild cats, pole cats, European mink and weasels. Infected animals particularly badgers, were most prevalent in the southernmost province of the BC (Araba) in areas dominated by arable land. Subsequent amplification and sequencing of a fragment of the rRNA internal transcribed spacer 2 (ITS2) from a subset of rtPCR positives samples confirmed the species as L. infantum, showing a high sequence homogeneity with ITS2 sequences of L. infantum from dogs and humans from southern Spain. In summary, this study reports for the first time L. infantum infection in wild carnivores from the BC including in stone martens, pole cats and minks in which infection has not been previously described. It supports the need to study infection in dogs and people in this region and is an example of the value of infection surveillance in wildlife to assess potential risks in the domestic environment and their role in spreading infections in non-endemic areas.

Microscopical and Immunological Features of Tuberculoid Granulomata and Cavitary Pulmonary Tuberculosis in Naturally Infected Goats

Caprine tuberculosis is caused by bacteria of the Mycobacterium tuberculosis complex (Mycobacterium bovis and Mycobacterium caprae). Although typical tuberculoid granulomata are usually observed in the lungs and lymph nodes of infected goats, the presence of cavitary lesions with exuberant mycobacterial growth is also a common feature in this species. The aim of this study was to characterize the immunological mechanisms that lead to liquefaction and cavity formation by comparing granulomata and cavitary lesions. Samples from animals positive by skin testing were collected for microscopical and immunohistochemical examination. Samples were also collected for analysis of cytokine gene expression in the lesions by real time polymerase chain reaction. There were marked differences between granulomata and cavitary lesions. In cavitary lesions there was a substantial population of neutrophils and a significant decrease in the number of CD4(+) T cells, with concomitant increases in other T-cell populations (CD8(+) and cells expressing the γδ form of the T-cell receptor). The enzyme iNOS was strongly expressed by macrophages in the cavitary lesions. There was no difference in the balance of pro- and anti-inflammatory cytokine gene expression in the lesions. These findings suggest that cavitary lesions are reactivation sites, where conditions are optimal for Mycobacterium proliferation and that immunological mechanisms may underlie the severe destruction of lung tissue that characterizes the cavitary pathology.

Protection conferred by commercially available vaccines against Chlamydophila abortus in a mouse model.

OVINE enzootic abortion (OEA), which is caused by Chlamydophila abortus (Chlamydia psittaci serotype 1), is an economically important disease in many countries. Inactivated vaccines prepared from egg-grown or cell cultures induce immunity in ewes and form the basis of products which have been in use for several decades. Although these vaccines have been commercially available for the prevention of OEA, it has been reported that apparent breakdowns appear every year in correctly vaccinated flocks (Aitken and others 1990). An alternative approach has been the development of

Chlamydophila abortus infection in the mouse: a useful model of the ovine disease.

Chlamydophila (C.) abortus is an obligate intracellular bacterium able to colonize the placenta of several species of mammals, which may induce abortion in the last third of pregnancy. The infection affects mainly small ruminants resulting in major economic losses in farming industries worldwide. Furthermore, its zoonotic risk has been reported in pregnant farmers or abattoir workers. Mouse models have been widely used to study both the pathology of the disease and the role of immune cells in controlling infection. Moreover, this animal experimental model has been considered a useful tool to evaluate new vaccine candidates and adjuvants that could prevent abortion and reduce fetal death. Future studies using these models will provide and reveal information about the precise mechanisms in the immune response against C. abortus and will increase the knowledge about poorly understood issues such as chlamydial persistence.

Differences in lymphocyte subpopulations from peripheral blood and lymphoid organs in natural caprine tuberculosis infection.

Although the cell-mediated immune response is known to be a critical factor in host defence against intracellular mycobacterial infection, the different components of the T-cell response are unclear, particularly in caprine infection. In this study we examine the differences in the lymphocyte population of peripheral blood, spleen and mediastinal and superficial lymph nodes in 11 naturally infected goats showing positive reactions in the comparative tuberculine intradermal test. According to the different types of lesion showing, the goats were classified into proliferative or exudative tuberculosis. The results obtained by fflow cytometry analysis indicated that the main differences in peripheral blood were in the CD4 T-cell population, which decreased markedly in goats with exudative tuberculosis, while the CD8 and B cells increased in number. The gamma/delta T cells did not show significant differences in either type of tuberculosis, while interleukin-2 receptor cells decreased slightly in the exudative tuberculosis. The CD4:CD8 ratio was higher than 1 in goats with proliferative tuberculosis and lower than 1 in goats with exudative tuberculosis. In general, the lymphoid organs of the goats with exudative tuberculosis showed a significant increase in the number of CD8 T cells (CD4:CD8 ratio of less than 1) whereas no significant differences were observed in the CD4 T population between either type of tuberculosis.