Bayesian Evaluation of Three Serological Tests for Detecting Antibodies against Brucella spp. among Humans in the Northwestern Part of Ecuador.

Brucellosis is an important but neglected zoonosis that causes serious economic losses both in livestock and human populations. The aim of the present study was to estimate the true prevalence of brucellosis together with diagnostic sensitivity and specificity of three serological tests in humans of the northwestern part of Ecuador using a Bayesian approach adjusted for the dependencies among the multiple tests to avoid any misinterpretation. In addition, the causal agent responsible for human brucellosis was also identified. Using a total of 3,733 samples collected from humans in this area between 2006 and 2008, the prevalence of human brucellosis and the diagnostic test characteristics of the Rose Bengal fast agglutination test (RBT), Wright's slow agglutination test with ethylenediaminetetraacetic acid disodium salt dehydrate (EDTA) (SAT-EDTA), and indirect ELISA (iELISA) were estimated using a Bayesian approach. The estimated true prevalence of human brucellosis was 1% (credibility interval: 0.4-1.6). The sensitivities of iELISA and RBT were higher than and similar (95.1% and 95.0%, respectively) to those of SAT-EDTA (60.8%). Even though all tests indicated a high specificity (> 99.0%), the specificity of SAT-EDTA was highest (99.9%). The circulating strain in this study area was identified to be Brucella abortus biotype 4 based on culture and microbiological characterization. The RBT and the iELISA are recommended for estimating the true prevalence of human brucellosis and/or for surveillance programs following their high sensitivities and specificities. The proposed strategy supports evidence-based medicine for clinicians and policy-makers to ensure appropriate preventive and control program of brucellosis worldwide.

The unexpected discovery of Brucella abortus Buck 19 vaccine in goats from Ecuador underlines the importance of biosecurity measures.

Very few, mostly old, and only preliminary serological studies of brucellosis in goats exist in Ecuador. In order to assess the current epidemiological situation, we performed a cross-sectional serological study in the goat populations of Carchi (n = 160 animals), Pichincha (n = 224 animals), and Loja provinces (n = 2024 animals). Only two positive serological results (RB negative and SAT-EDTA ≥400 IU/ml) were obtained in lactating goats from the same farm in Quito (Pichincha province). Additionally, milk was sampled from 220 animals in Pichincha province. The present study indicates a low apparent prevalence in Pichincha province and absence in Carchi and Loja provinces. A total of 25 positive milk ring tests (MRT) were obtained in Pichincha province yielding a prevalence of MRT of 11.16%. Subsequent culture was performed on the positive MRT samples. All results were negative, apart from a single sample, obtained from a serologically positive goat in Quito, that was positive for Brucella abortus strain 19 (B19). Several hypotheses are forwarded concerning this unexpected result. The most likely hypothesis is the possible accidental use of a needle, previously used for vaccination of cattle with the said vaccine, for the administration of drug treatment to the goat. This hypothesis underlines the necessity of biosecurity measures to prevent this type of accidents.

Long-term assessment of glucosuria in captive okapi (Okapia johnstoni) after a dietary change.

Glucosuria in okapis (Okapia johnstoni) was first documented in 1980, yet the etiology remains unclear. In August 2006, an attempt to lower glucosuria in captive okapi by diet modification (omitting all fruit and adding unmolassed beet pulp) was started at the Antwerp Zoo. To study the possible relationship between glucosuria and diet, stress, and/or pregnancy, four okapis were monitored over a period of 4.5 yr. One animal, born in 2006, became glucosuric near the age of three. Three okapis were adults at the start of the study and had been glucosuric for more than 5 yr. The glucose/creatinine urinary ratio values of these four glucosuric animals did not change considerably over time despite dietary changes. Stress did not appear to influence glucosuria in these okapi. Urinary ratio decreased during the second half of pregnancy in two females. In conclusion, the diet change did not reduce glucosuria, but pregnancy appeared to lower urinary glucose in okapis.

Human brucellosis in northwest Ecuador: typifying Brucella spp., seroprevalence, and associated risk factors.

Human brucellosis in Ecuador is underreported and based only on passive surveillance. Since 2008, brucellosis was removed from the list of communicable diseases in the country. Until now, the true human brucellosis picture has not yet been determined. The aim of this study was to determine the seroprevalence of the disease, identify risk factors associated with brucellosis seropositivity in humans, and isolate circulating strains of Brucella spp. in the northwestern part of Ecuador. Between 2006 and 2008, a large transect survey was conducted, based on blood sampling of people from the northwestern part of Ecuador (n=3733) together with an epidemiological inquiry. On the basis of three diagnostic tests used in parallel, the overall seroprevalence was estimated as 1.88% (95% confidence interval [CI] 1.48-2.38). Based on a multivariable random effects logistic regression analysis, the main risk factors associated with human brucellosis seropositivity were contact with livestock (odds ratio [OR]=3.0; CI 1.25-7.08), consumption of fetus and placenta (OR=2.5; CI 1.18-5.22), and involvement in activities at risk for brucellosis infection (OR=1.8; CI 1.00-3.35). Noticeable variation in brucellosis seropositivity among humans within cantons was observed. The circulating strain was Brucella abortus biotype 4. This study emphasized that contact with livestock, consumption of fetus and placenta, and occupational hazard group were all significant risk factors for the transmission of brucellosis among individuals in the northwestern part of Ecuador. Alongside encouraging the launching of educational campaigns against brucellosis, especially in rural areas where 36% of the population lives, controlling this zoonotic disease in animals will directly benefit its prevention in humans, especially because there is no safe and efficacious vaccine against brucellosis in humans.

First report of orchitis in man caused by Brucella abortus biovar 1 in Ecuador.

We present a 44-year-old man from a rural community in northern Ecuador who worked on a cattle farm where he was involved with primary veterinary care, including assistance during births (or calving) and placenta retention and artificial insemination, with minimal precautions. In September of 2009, quite abruptly, he developed asthenia and hypersomnia without any apparent cause or symptoms like fever, chills, or night sweats. On November 14, 2009, he suffered from pain and edema in the right testicle that coincided with pain in the abdomen. Clinical, serological, and bacteriological investigations confirmed the first case of unilateral orchitis in man in Ecuador caused by Brucella abortus biovar 1. Because brucellosis is a neglected disease, special attention should be given to it in the training of medical and veterinary students.

Prevalence of methicillin-resistant Staphylococcus aureus in mammals of the Royal Zoological Society of Antwerp, Belgium.

Contrary to the numerous reports on methicillin-resistant Staphylococcus aureus (MRSA) in domestic animals, only three articles concerning zoo animals are documented in the literature. A skin infection of an African elephant (Loxodonta africana) calf was most likely acquired from an infected caretaker. Another zoo detected MRSA in the rumen content of a mouflon (Ovis aries), and, in a third facility, it was reported in a fistulous wound at the coronary band of a digit of an Indian rhinoceros (Rhinoceros unicornis). In the present study, which lasted 13 months and involved 93 different individual mammals that belonged to 40 species and 19 families housed in the Royal Zoological Society of Antwerp, Belgium, this study reports the absence of MRSA in swabs of nostrils, skins, conjunctiva, vulva, abscess, and arm rests in public spaces. Samples were enriched overnight and inoculated on a selective chromogenic medium.

Validation of an immunohistochemical assay for bovine cysticercosis, with comparison to a standard histological method.

The larval stage (syn Cysticercus bovis) of the human tapeworm Taenia saginata causes cysticercosis in cattle, which has both aesthetic and food safety implications to consumers of beef. A monoclonal antibody-based immunohistochemical (IHC) assay developed to improve postmortem diagnosis of this parasite and a standard histological method were assessed to determine their fitness for intended use. Sections from 169 known-positive specimens of T. saginata from experimentally or naturally infected cattle, and from 30 known-negative specimens and lesions of various etiologies from non-infected cattle, were tested. The IHC assay identified significantly more known positive bovine cysticerci than the histological method (91.7% and 38.5%, respectively). Positive IHC staining occurred on sections from other cestode species, but should not affect the diagnostic specificity of this assay for bovine cysticercosis, due to the different host and/or tissue preferences amongst these parasites. Use of the IHC assay should improve the reliability of diagnosing lesions caused by degenerated cysticerci, facilitating more effective and efficient control of bovine cysticercosis.

Specific detection and identification of African trypanosomes in bovine peripheral blood by means of a PCR-ELISA assay.

The aim of the present study was to develop a PCR-ELISA assay for the detection and differentiation of the main African pathogen trypanosomal species present in peripheral blood of cattle. The proposed methodology allows to specifically differentiate Trypanosoma congolense, Trypanosoma vivax and the subgenus Trypanozoon, by means of a sensitive universal PCR amplifying trypanosome DNA followed by an ELISA-based hybridization with three highly specific probes. The semi-nested PCR had a sensitivity of 15 fg, 15 fg, and 0.15 fg of DNA from T. vivax, T. congolense, and Trypanosoma brucei brucei, respectively that is sufficient to detect parasites in blood during the chronic phase of the disease. Biotinylated second round asymmetric PCR amplification products were used in an ELISA set up using three species-specific probes for the diagnosis of T. congolense (type Riverine, Kilifi or Savannah), T. vivax and T. brucei brucei. A factor O.D. of 0.082 was determined on blood samples from bovines (n=18) from a non-endemic area in Africa. In a pilot study of blood samples of naturally and experimentally Trypanosoma infected cattle previously characterized by PCR-RFLP (n=42), a high rate of concordance (93.3%) was found between PCR-RFLP and PCR-ELISA. There is a good ratio between positive and negative O.D. values (3.00 vs. 0.1) and the technique can also be used to distinguish mixed infections.

Detection of Taenia solium antigens and anti-T. solium antibodies in paired serum and cerebrospinal fluid samples from patients with intraparenchymal or extraparenchymal neurocysticercosis.

BACKGROUND: Neurocysticercosis (NCC) is a frequent cause of epilepsy worldwide. Compared with the more common parenchymal brain cysts, extraparenchymal infections are difficult to manage and have a poor prognosis. Serological assays are used to detect circulating Taenia solium antigens or anti-T. solium antibodies in serum or cerebrospinal fluid (CSF) samples. There are no guidelines on whether to use serum or CSF specimens for a particular assay. METHODS: We obtained paired serum and CSF samples from 91 patients with NCC (48 had intraparenchymal NCC, and 43 had extraparenchymal NCC) for detection of antibodies, using an enzyme-linked immunotransfer blot (EITB) assay, and antigens, using a monoclonal antibody-based enzyme-linked immunosorbent assay (ELISA). RESULTS: For the intraparenchymal NCC group, the EITB assay yielded more true-positive results for serum samples, and the ELISA yielded slightly more true-positive results for CSF samples than for serum samples, but none of these differences were statistically significant. Most patients with calcified NCC were antibody positive but antigen negative. For extraparenchymal disease, all samples were antibody positive, and all but 2 were antigen positive, with most samples containing high antigen levels. CONCLUSIONS: The sensitivity of antibody-detecting EITB assays is not increased through the use of CSF samples rather than serum samples. The antigen-detecting ELISA performed better for CSF samples than for serum samples, but for both specimen types it was less sensitive than the EITB assay. Active and inactive NCC are better differentiated from each other by the antigen-detecting ELISA, for both serum and CSF samples. High antigen levels suggest the presence of subarachnoid NCC.

Urine antigen detection for the diagnosis of human neurocysticercosis.

Neurocysticercosis (NCC) is a major cause of seizures and epilepsy. Diagnosis is based on brain imaging, supported by immunodiagnosis in serum or cerebrospinal fluid (CSF). Lumbar puncture is invasive and painful. Blood sampling is slightly painful and poorly accepted. Urine antigen detection has been used for other parasites and tried in NCC with suboptimal performance. We used a monoclonal antibody-based ELISA to detect Taenia solium antigens in urine from 87 Peruvian neurocysticercosis patients (viable cysts, N = 34; subarachnoid cysticercosis, N = 10; degenerating parasites, N = 7; calcified lesions, N = 36) and 32 volunteers from a non-endemic area of Peru. Overall sensitivity of urine antigen detection for viable parasites was 92%, which decreased to 62.5% in patients with a single cyst. Most patients (30/36, 83%) with only calcified cysticercosis were urine antigen negative. Antigen levels in paired serum/urine samples (evaluated in 19 patients) were strongly correlated. Non-invasive urine testing for T. solium antigens provides a useful alternative for NCC diagnosis.

Validation of meat inspection results for Taenia saginata cysticercosis by PCR-restriction fragment length polymorphism.

Bovine cysticercosis is a zoonosis caused by the larval stage (cysticercus) of the human tapeworm Taenia saginata. Infected cattle is an important food safety issue besides an economic concern. Humans get infected by eating raw or undercooked meat containing viable cysticerci. Visual meat inspection of bovines is the only public health measure implemented to control transmission to humans, but it lacks sensitivity and objectivity. It may underestimate the prevalence of the disease by a factor 3 to 10. Furthermore, the success of the method depends on the expertise of the meat inspector as well as on the stage of development of the cysticerci. The focus of this study was to develop and explore the usefulness of a PCR assay as an objective alternative to evaluate the meat inspector's visual inspection results. Hereto, a PCR was developed for the detection of T. saginata DNA in muscle lesions. Based on the laboratory classification of lesions, almost 97% of viable cysts were confirmed by PCR, while for dead cysts, the percentage was approximately 73%. Taken together, these data demonstrate the difficulties of visual meat inspection and their objective interpretation, emphasizing the need to improve current assays to strengthen the control of bovine cysticercosis.

Preliminary observations on Mycobacterium spp. in dairy cattle in Ecuador.

This study evaluated bovine tuberculosis in Mejia canton, a major dairy cattle production region in Ecuador. Randomly selected cattle (1,012 from 59 farms) classified according to herd size were tested by the single tuberculin test (STT). Sixty days later, positive reactors were tested again by the comparative tuberculin test (CTT). In addition, tissue samples from two STT-CTT-positive reactors detected on a farm were obtained in a local slaughterhouse and analyzed bacteriologically. A total of 4.24% of the cattle were positive in the STT and 3.85% were positive in the CTT, with the highest number (7.95%) in large herds versus 3.4% in medium herds and 0.3% in small herds. Mycobacterium bovis was isolated from mesenteric lymph nodes and lungs of one animal. A 16S ribosomal RNA-based polymerase chain reaction confirmed culture results and differentiated mycobacteria other than M. tuberculosis. This study confirms the zoonotic importance of tuberculosis in Ecuadorian dairy cattle with herd size likely to be a crucial parameter in the prevalence of the disease. The implementation of a national control program is necessary and should be based on the detection of positive cattle by STT in combination with CTT.

Reactive oxygen species and 12/15-lipoxygenase contribute to the antiproliferative capacity of alternatively activated myeloid cells elicited during helminth infection.

Understanding the role of CD11b(+)GR-1(+) myeloid suppressor cells in the immune suppression and immunoregulation associated with a variety of diseases may provide therapeutic opportunities. In this article, we show, in a model of helminth infection, that CD11b(+)GR-1(+) myeloid suppressor cells but not CD11b(+)F4/80(high) mature macrophages expanded in the peritoneal cavity of BALB/c mice implanted with Taenia crassiceps. Peritoneal cell populations from early stage-infected animals impaired T cell proliferation by secreting NO. Yet, they lost their ability to secrete NO in the late stage of infection. Concomitantly, their capacity to exert arginase activity and to express mRNAs coding for FIZZ1 (found in inflammatory zone 1), Ym, and macrophage galactose-type C-type lectin increased. Furthermore, cells from early stage-infected mice triggered T cells to secrete IFN-gamma and IL-4, whereas in the late stage of infection, they only induced IL-4 production. These data suggest that CD11b(+)GR-1(+) myeloid suppressor cells displaying an alternative activation phenotype emerged gradually as T. crassiceps infection progressed. Corroborating the alternative activation status in the late stage of infection, the suppressive activity relied on arginase activity, which facilitated the production of reactive oxygen species including H(2)O(2) and superoxide. We also document that the suppressive activity of alternative myeloid suppressor cells depended on 12/15-lipoxygenase activation generating lipid mediators, which triggered peroxisome proliferator-activated receptor-gamma. IL-4 and IL-13 signaling contributed to the expansion of myeloid suppressor cells in the peritoneal cavity of T. crassiceps-infected animals and to their antiproliferative activity by allowing arginase and 12/15-lipoxygenase gene expression.

Macrophage galactose-type C-type lectins as novel markers for alternatively activated macrophages elicited by parasitic infections and allergic airway inflammation.

Molecular markers, especially surface markers associated with type II, cytokine-dependent, alternatively activated macrophages (aaMF), remain scarce. Besides the earlier documented markers, macrophage mannose receptor and arginase 1, we demonstrated recently that murine aaMF are characterized by increased expression of found in inflammatory zone 1 (FIZZ1) and the secretory lectin Ym. We now document that expression of the two members of the mouse macrophage galactose-type C-type lectin gene family (mMGL1 and mMGL2) is induced in diverse populations of aaMF, including peritoneal macrophages elicited during infection with the protozoan Trypanosoma brucei brucei or the Helminth Taenia crassiceps and alveolar macrophages elicited in a mouse model of allergic asthma. In addition, we demonstrate that in vitro, interleukin-4 (IL-4) and IL-13 up-regulate mMGL1 and mMGL2 expression and that in vivo, induction of mMGL1 and mMGL2 is dependent on IL-4 receptor signaling. Moreover, we show that expression of MGL on human monocytes is also up-regulated by IL-4. Hence, macrophage galactose-type C-type lectins represent novel surface markers for murine and human aaMF.

Bovine cysticercosis: preliminary observations on the immunohistochemical detection of Taenia saginata antigens in lymph nodes of an experimentally infected calf.

A newly developed immunohistochemical test was used for the first time to demonstrate the presence of Taenia saginata (Cysticercus bovis) antigens in the lymph nodes of a heifer calf experimentally inoculated with Taenia saginata eggs. The new test should aid in the differential diagnosis of eosinophilic lymphadenitis in cattle.

Diagnosis of taenia saginata cysticercosis by immunohistochemical test on formalin-fixed and paraffin-embedded bovine lesions.

A new method of diagnosing cysticercus or larval stage of the human tapeworm, Taenia saginata, also known as Cysticercus bovis, in formalin-fixed bovine tissue was developed using a monoclonal antibody to T. saginata and avidin-biotin complex immunohistochemistry. Grossly recognizable viable and degenerate cysts were identifiable after immunohistochemical staining and could be differentiated from Sarcocystis, Actinobacillus, or non-cyst, normal bovine structures. Thenew test should permit laboratory confirmation of suspected T. saginata cysticercus lesions.

Merogony in in vitro cultures of Theileria parva.

In vitro studies were focussed on the duration and cessation of merogony in Theileria parva infected blood lymphocyte cell cultures. The cultures were infected using purified tick stabilates as an alternative to in vitro infections, using sporozoites obtained by labour intensive dissections of salivary glands from infected ticks. After establishment of infection in peripheral blood lymphocytes (PBL), merozoites were temporarily produced for about 2 months after which lymphoblasts only contained schizonts.

Regional status, epidemiology and impact of Taenia solium cysticercosis in Western and Central Africa.

In West Africa, Taenia solium cysticercosis in both pigs and man has been reported in Benin, Burkina-Faso, Ghana, Ivory Coast, Senegal and Togo, and although official data are lacking, T. solium is anticipated to be present in most of the pig-raising regions of other West African countries as well. In some regions of Nigeria, the prevalence of porcine cysticercosis and human taeniosis is quite high (20.5 and 8.6%, respectively). Surprisingly, however, no cases of human cysticercosis have been reported, although epilepsy is very common. Large epidemiological surveys have only been carried out in Togo and Benin, where the prevalence of human cysticercosis was 2.4 and 1.3%, respectively. In Central Africa, porcine and human cysticercosis are (hyper)-endemic in Rwanda, Burundi, the Democratic Republic of Congo and Cameroon. The parasite also has been reported in pigs in Chad and Angola. Cysticercosis has been shown to be one of the major causes of epilepsy in Cameroon with figures as high as 44.6%. Cameroon is one of the few countries where the taeniosis-cysticercosis complex has been examined more in detail. In the Western province of Cameroon large scale surveys have shown that active cysticercosis is present in 0.4-3% of the local population and in 11% of the village pigs. However, the prevalence of adult T. solium was only 0.1%, which underscores the frequency of the T. solium paradox. Based on the available information, a very conservative economic estimate indicates that the annual losses due to porcine cysticercosis in 10 West and Central African countries amount to about 25 million Euro. The financial losses due to human cysticercosis are very difficult to estimate, but are certainly exceeded by the social impact of the disease, especially because of the particular perception of epilepsy in many African communities. It is concluded that the true prevalence of T. solium cysticercosis in pigs and humans in Central and West Africa remains underestimated because of unreliable slaughterhouse data and the lack of awareness and diagnostic facilities in the public health sector.

Immunodiagnostic tools for human and porcine cysticercosis.

The development of improved immunodiagnostic tools has contributed to our knowledge on the importance of taeniosis/cysticercosis by enabling sero-epidemiological surveys and community-based studies to be carried out. Immunodiagnostic techniques include detection methods for specific antibodies and for circulating parasite antigen in serum or cerebrospinal fluid. The antigens used in immunoblot and enzyme-linked immunosorbent assay (ELISA) for antibody detection have evolved from crude extracts to highly purified specific fractions and recombinant antigens of the glycoprotein family, increasing both the sensitivity and the specificity of the tests. The application of ELISA for the detection of circulating parasite antigens may present some diagnostic advantages since it demonstrates not only exposure but also active infections. Until now only a few of the current techniques have been standardised and fully validated, making comparisons between studies difficult. The lack of a gold standard is a serious drawback. In surveys on cysticercosis, antibody detection systems have been useful in identifying the risk factors associated with transmission of Taenia solium; a high seroprevalence in a community indicates a "hot spot" where preventive and control measures should be applied. In contrast, the potential use of immunodiagnostic tools to identify cases of neurocysticercosis (NCC) in man is subject to debate. The correlation between a positive serology and neurological symptoms and/or lesions indicative for NCC on neuro-imaging techniques is poor to fair in most studies. This may be explained by the unpredictable clinical outcome of the infection and the variable immunological response of the human host to infection. A major problem is that in many developing countries, neuro-imaging methods are inaccessible and/or too expensive for the rural population at risk. Under these conditions, serology may provide the only tool for diagnosis of the infection.