New and rapid strategies for the diagnosis of bovine paratuberculosis "in situ" using latex particles.

The chemical coupling of a protoplasmatic antigen from Mycobacterium avium subsp. paratubeculosis onto core-shell carboxylated particles was investigated with the aim of producing latex-protein complexes to be used in immunoagglutination assays capable of detecting bovine paratuberculosis disease. For this purpose, sensitizations were carried out using both colored and not colored carboxylated latexes as well as the protoplasmatic antigen at pH close to its isoelectric point to favor the antigenic protein to approach the particle surface. In all cases, higher fractions of proteins were chemically-bound to carboxyl groups on the surface of the particles. The assessment of the performance of the visual immunoagglutination assays consisted of evaluating 111 sera from healthy and infected bovines with Mycobacterium avium subsp. paratuberculosis. Complexes obtained from the colored latex allowed an acceptable visual discrimination between the studied positive and negative sera. Most of the positive samples showed strong to very strong agglutination and only a few samples reacted weakly, i.e. a sensitivity of 70%. The specificity of the assay, on the other hand, was 86%. Therefore, this rapid detection technique allows an easy and inexpensive identification of animals possibly infected with paratuberculosis "in situ" in the herds.

Study on prevalence and liver function test enzymes of differently plumaged peafowls (Pavo cristatus) infected with Toxoplasma gondii in captivity / [Estudo sobre enzimas de prevalência e teste da função hepatica de pavões de plumage diferente (Pavo cristatus) infectados com Toxoplasma gondii em cativeiro]

O presente estudo foi realizado para determinar a prevalência geral de toxoplasmose em pavões de plumagem diferente e seu efeito nas enzimas de teste da função hepática dos hospedeiros. Um total de cem pavões de plumas diferenciais, como ombro preto (n = 52), azul (n = 28), branco (n = 10) e arlequim (n = 10) foram estudados no zoológico de Bahawalpur, no Paquistão, usando o Latex Agglutination Test (LAT) e ensaio imunossorvente ligado a enzima (ELISA). A prevalência geral por LAT e ELISA foi de 37% e 30%, respectivamente. Por LAT, observou-se uma prevalência não significativamente maior (P≥0,05) em gênero (37,77%) nos machos do que nas fêmeas (36,36%), enquanto os adultos apresentaram uma prevalência maior (37,97%) em relação aos jovens (33,33%). De acordo com o ELISA, uma prevalência significativamente (P <0,05) maior (35,55%) foi observada nos machos do que nas fêmeas (25,45%) e significativamente (P <0,05) maior prevalência (31,64%) foi registrada nos adultos do que nos jovens (23,80%). A análise do perfil bioquímico sérico mostrou que o nível de bilirrubina não teve elevação significativa nos hospedeiros infectados, em comparação aos não infectados, enquanto a concentração de albumina, alanina aminotransferase (ALT), aspartato aminotransferase (AST), fosfatase alcalina (ALP) foi significativamente (P <0,05) diferente nos hospedeiros infectados. Conclui-se que a toxoplasmose afeta as enzimas do teste da função hepática. Essa é uma pesquisa preliminar e requer mais pesquisas em todo o país, com populações e amostras maiores.(AU)

Development of a simple and economical diagnostic test for canine leishmaniasis.

Visceral leishmaniasis is a public health problem worldwide. The early diagnosis in dogs is crucial, since they are an epidemiologically relevant reservoir of the disease. The aim of a field study is to early identify the disease allowing rapid intervention to reduce its effects. We propose an immunoagglutination test as a visual in situ method for diagnosis of canine visceral leishmaniasis. Latex-protein complexes were sensitized by covalent coupling of a chimeric recombinant antigen of Leishmania spp. onto polystyrene latex with carboxyl functionality. The reaction time and the antigen concentration under which the immunoagglutination assay shows greater discrimination between the responses of a positive control serum and a negative control serum were determined. Then, the latex-protein complexes were evaluated as a visual diagnostic tool with a panel of 170 sera. The test may be read between 2 and 5 min and can be performed even using sera with elevated concentration of lipids, bilirubin or with variable percentage of hemolysis. The sensitivity, the specificity and the diagnostic accuracy were 78%; 100% and >80%, respectively. The visual immunoagglutination test is of potential application as a method for field studies because it shows results in less than 5 min, it is easy to implement and does not require sophisticated equipment.

Analysis of bovine rotavirus strains circulating in diarrheic dairy calves in Uberaba, Minas Gerais, Brazil, during 2008-2009 / Análise de cepas de rotavírus bovino circulantes em bezerros leiteiros com diarreia em Uberaba, Minas Gerais, Brasil, durante 2008-2009

O objetivo deste estudo foi detectar rotavírus em fezes de bezerros com diarreia em Uberaba, MG, e caracterizar os genes VP7 e VP4 por meio da genotipagem e da análise filogenética. Setenta e quatro amostras foram coletadas entre novembro de 2008 e setembro de 2009. A detecção do vírus foi feita por teste de aglutinação e as amostras positivas foram submetidas à transcrição reversa, seguida de reação em cadeia da polimerase (RT-PCR), tipagem por PCR e sequenciamento. A taxa de detecção de rotavírus foi de 6,8% e todas as amostras apresentaram o genótipo G6P[5]. A análise filogenética mostrou que as amostras do genótipo G6 pertencem à linhagem IV e que, para ambos os genes (VP7 e VP4), as amostras deste estudo compõem um sub-cluster à parte daquele das cepas referências e das amostras campo mais similares. O alinhamento das sequências de aminoácidos deduzidas mostrou substituições em regiões antigênicas quando comparadas com as sequências das cepas bovinas UK e NCDV, presentes nas vacinas disponíveis no Brasil. Uma nova sublinhagem genética de G6P[5] foi evidenciada neste estudo. Substituições de aminoácidos nas regiões antigênicas dos rotavírus e a circulação de novas variantes podem representar desafios para as vacinas utilizadas atualmente. O presente estudo contribui para a compreensão da epidemiologia dos rotavírus bovinos no Brasil.(AU)

Analysis of bovine rotavirus strains circulating in diarrheic dairy calves in Uberaba, Minas Gerais, Brazil, during 2008-2009 / Análise de cepas de rotavírus bovino circulantes em bezerros leiteiros com diarreia em Uberaba, Minas Gerais, Brasil, durante 2008-2009

O objetivo deste estudo foi detectar rotavírus em fezes de bezerros com diarreia em Uberaba, MG, e caracterizar os genes VP7 e VP4 por meio da genotipagem e da análise filogenética. Setenta e quatro amostras foram coletadas entre novembro de 2008 e setembro de 2009. A detecção do vírus foi feita por teste de aglutinação e as amostras positivas foram submetidas à transcrição reversa, seguida de reação em cadeia da polimerase (RT-PCR), tipagem por PCR e sequenciamento. A taxa de detecção de rotavírus foi de 6,8% e todas as amostras apresentaram o genótipo G6P[5]. A análise filogenética mostrou que as amostras do genótipo G6 pertencem à linhagem IV e que, para ambos os genes (VP7 e VP4), as amostras deste estudo compõem um sub-cluster à parte daquele das cepas referências e das amostras campo mais similares. O alinhamento das sequências de aminoácidos deduzidas mostrou substituições em regiões antigênicas quando comparadas com as sequências das cepas bovinas UK e NCDV, presentes nas vacinas disponíveis no Brasil. Uma nova sublinhagem genética de G6P[5] foi evidenciada neste estudo. Substituições de aminoácidos nas regiões antigênicas dos rotavírus e a circulação de novas variantes podem representar desafios para as vacinas utilizadas atualmente. O presente estudo contribui para a compreensão da epidemiologia dos rotavírus bovinos no Brasil.(AU)

Diversity of Staphylococcus species and prevalence of enterotoxin genes isolated from milk of healthy cows and cows with subclinical mastitis.

The objectives of this study were to determine the occurrence and diversity of Staphylococcus spp. in milk from healthy cows and cows with subclinical mastitis in Brazil and to examine the profile of enterotoxin genes and some enterotoxins produced by Staphylococcus spp. A total of 280 individual mammary quarter milk samples from 70 healthy cows and 292 samples from 73 cows with subclinical mastitis were collected from 11 farms in the state of São Paulo, Brazil. Staphylococcus spp. were recovered from 63 (22.5%) samples from healthy cows and from 80 samples (27.4%) from cows with mastitis. The presence of Staphylococcus aureus was significantly different between these 2 groups and was more prevalent in the cows with mastitis. The presence of Staphylococcus saprophyticus was also significantly different between these 2 groups, but this organism was more prevalent in healthy cows. No statistically significant differences were observed in the numbers of other staphylococci in milk samples from the 2 groups. The sea gene was the most prevalent enterotoxin gene in both groups. Eight of 15 (53.3%) Staph. aureus carried this gene and all produced the SEA toxin. In the coagulase-negative staphylococci (CNS) group, 61 of 128 (47.5%) had the same gene and just 1 (1.6%) Staphylococcus epidermidis strain produced the enterotoxin in vitro. Because CNS were isolated from both groups of cows and most CNS contained enterotoxin genes but did not produce toxins, the role of CNS in mastitis should be carefully defined.

Evaluation of a latex agglutination kit for detecting rotavirus in piglets

Background: The group A rotavirus is the most important ethiological agent that causes diarrhea in newborn humans and animals. They belong to the Reoviridae family and have a genome consisting of 11 segments of double-stranded RNA enclosed in a triple-layered capsid. Rotaviruses are classified in six groups (A to G) based on the VP6 capsid protein or on the migration pattern of genomic segments in polyacrylamide gel. Groups A, B, and C were found either in humans or in animals, while groups D to G were found only in animals. The outer layer is formed by two proteins, VP7 and VP4, which elicit neutralizing antibody responses and form the basis of the current dual classification system in G (VP7) and P (VP4) types. The early diagnostic of this viral infection in raising farms is crucial for preventing the dissemination of the disease among the animals. Latex agglutination tests (LAT) are quick and easily done, and require low financial investments. Materials, Methods & Results: The present study evaluated the Rotavirus Latex® kit (Richmond Immunosystems Diagnostics) currently used with humans for detecting the presence of rotavirus antigen in diarrheic feces of piglets. In order to confirm the results obtained with the LAT, samples were analyzed by EIA with the Ridascreen rotavirus® kit (R-Biopharm). The results were compared with the rotavirus determination test by polyacrylamide gel electrophoresis (PAGE). During March 2007 and March 2008, 328 excrement samples of diarrheic piglets were collected at a hog-raising farm in the state of Rio Grande do Sul, Brazil. Results showed that the rotavirus was present in 38 samples (11.6%) assayed with LAT, while PAGE detected the virus in 26 samples (7.9%). This means that 15 positive cases (4.6%) were not identified by PAGE. This difference shows the higher sensitivity of LAT against PAGE. LAT showed 88.5% of sensitivity, 95% of specificity, 60.5% of positive predictive value, 99% of negative predictive value and 94.5% of accuracy. Discussion: Latex agglutination is easy to perform in a short time and does not require expensive equipment or skilled personnel, and the reagents have long shelf lives. These factors make the LAT suitable and highly efficient for use in a clinical laboratory and a small farm as a rapid screening test for piglet rotavirus. A test used for detecting the etiological agent of gastroenteritis should be fast, easy to use, and specific for anticipating the disease treatment, thus minimizing unnecessary expenses and establishing prophylactic measures to protect the entire flock. The most important factor in choosing the method is the number of samples that should be collected, the qualification of the analysis laboratory, and the type of pathogen that has contaminated the flock. The diagnostic methods most easily applied are based on detecting viral particles or RNA from fecal samples. Most kits available in the market for detecting rotaviruses are designed for humans and are not adequate for use in veterinary diagnosis. Therefore, the results obtained herein presented high sensitivity and specificity for LAT, showing to be a valuable tool for diagnosing rotaviruses in pig feces. This system used in clinical laboratories might also be used in intensive animal farming systems or by small-scale pig raisers in a cooperative system.

Aspects of the natural history and virulence of S. agalactiae infection in Nile tilapia.

Streptococcus agalactiae is an emerging pathogen in Nile tilapia (Oreochromis niloticus) worldwide. To investigate aspects of the epidemiology, transmission and virulence of S. agalactiae infections, nine outbreaks of meningoencephalitis and septicemia in Nile tilapia farms in Brazil were analyzed. Records from the outbreaks revealed large variation in the weight of fish affected, high mortality, and disease occurrence at water temperatures above 26 degrees C. S. agalactiae was isolated from diseased fish from all farms, and 29 strains were identified by phenotypic tests and 16S rRNA gene sequencing. Five strains from different geographic origins were selected to determine the 50% lethal dose (LD(50)). All strains were highly virulent; for example, strain SA 20-06 had an LD(50) of 90 bacteria. To investigate S. agalactiae transmission, we conducted cohabitation assays with diseased and healthy fish and fish challenges using an immersion bath or gill inoculation. Strain SA 20-06 was used in all assays. The disease was reproduced with characteristic clinical signs and S. agalactiae was reisolated in all trials. The infection route studies were identified as by direct contact or through the water. In conclusion, S. agalactiae, a major pathogen of Nile tilapia in Brazil, exhibited high virulence, regardless of the geographic origin of the isolated strains.

Epizootic cutaneous pythiosis in beef calves.

Enzootic bovine granulomatosis (EBG) is a disease that affects beef calves in the flooded Savanna regions of Venezuela. Although Pythium insidiosum was originally the suspected etiologic agent, nothing was done to demonstrate its presence in the infected animals. The objective of this study was to investigate the etiologic agent causing cutaneous lesions in a group of 63 calves diagnosed with EBG. The collected samples were analyzed by histopathology, immunohistochemistry, serology, and mycological techniques. The clinical manifestations of BEG included ulcerated and swollen granulomatous lesions on the limbs of young calves. Histopathological studies revealed an eosinophilic inflammatory reaction with neutrophils, giant cells and branching hyphae with the Splendore-Hoeppli phenomenon. Immunofluorescence analysis identified the branching hyphae as elements of the fungal-like organism P. insidiosum. Wet mounts in 10% KOH revealed hyaline slender branching hyaline coenocytic hyphae, but cultures from those samples were negative. Anti-P. insidiosum immunoglobulins were also demonstrated in the collected sera of the affected bovines using latex agglutination, immunodiffusion, ELISA, and Western Blot. The histopathological, immunohistochemical, and serological tests showed for the first time that P. insidiosum could be involved in epizootic bovine pythiosis.

Seroprevalence of Toxoplasma gondii in American free-ranging or captive pumas (Felis concolor) and bobcats (Lynx rufus).

Toxoplasma gondii is a major zoonotic agent infecting a wide range of mammals, including wild felids. Like domestic cats, wild felids are involved in the complete infective cycle of T. gondii, as they can host in their gastrointestinal tract sexually mature parasites and shed infective oocysts in their feces. In order to evaluate the importance of this wildlife reservoir, 438 serum samples collected between 1984 and 1999 from 438 pumas (Felis concolor) and from 58 bobcats (Lynx rufus) from North America, Central America and South America were screened for antibodies to T. gondii. The overall prevalence of T. gondii antibodies was 22.4% in pumas and 51.7% in bobcats, with regional variations. Adults were more likely to be seropositive than juveniles and kittens (prevalence ratio (PR) = 2.61; confidence interval (CI) = 1.15, 4.04). In the US, pumas from the southwestern states (Arizona, California and New Mexico) were more likely to be seropositive for T. gondii ( PR = 2.61; 95% CI = 1.32-5.18 ) than pumas from the northwestern and mountain states (Colorado, Idaho, Oregon, Utah and Wyoming). Male pumas from the US were more likely to be seropositive than females (PR = 2.08; 95% CI = 1.11-3.92), whereas female pumas from Mexico, Central America and South America were more likely to be seropositive than female pumas from Canada and the US (PR = 2.49; 95% CI = 1.09-5.69). Captive pumas were also more likely to be seropositive (21.7%, 29/92) for T. gondii than free-ranging animals (19.9%, 69/346) (PR = 1.85; 95% CI = 1.06, 3.17).

Seroepidemiology of Toxoplasma gondii in dogs in Trinidad and Tobago.

A cross-sectional study was conducted to determine the seroprevalence of Toxoplasma gondii agglutinins and to investigate the relationship between various risk factors and occurrence of toxoplasmosis in dogs in Trinidad. Of a total 250 dogs, comprising domestic, hunting and stray dogs, 80 (32.0%) were positive for T. gondii agglutinins at a titre of > or =1:32 using a latex agglutination test. Stray dogs (60.5%) had statistically significantly higher (P<0.001) seroprevalence for toxoplasmosis than hunting dogs (30.5%) and domestic dogs (25.5%). Amongst dogs whose ages were known, the prevalence of toxoplasmosis was significantly highest (P=0.037) in dogs in the >2-3 years age group compared with other age groups. Dogs that consumed home-cooked foods had a seroprevalence of 32.9% compared with those fed commercial dog foods (17.2%) and dogs fed both home-cooked and commercial foods (21.0%). However, the difference was not statistically significant (P>0.05; chi(2)). The rather high prevalence of toxoplasmosis in stray dogs is a good indication of the extent of the infection in the environment.

Diagnosis of equine piroplasmosis in Brazil by serodiagnostic methods with recombinant antigens.

Serum samples from horses in the States of Sao Paulo and Mato Grosso do Sul, Brazil were examined for diagnosis of equine piroplasmosis by both the latex agglutination test (LAT) and enzyme-linked immunosorbent assay (ELISA) with recombinant antigens. Of the 47 samples analyzed, 38 (81%) and 42 (90%) samples were positive for B. equi infection and B. caballi infection, respectively. In addition, 35 (75%) samples were positive for both B. equi and B. caballi infections. These results indicate that equine piroplasmosis is widespread and therefore a cause for serious concern in the States of Sao Paulo and Mato Grosso do Sul, Brazil.

Antibody levels in goats fed Toxoplasma gondii oocysts.

Outbred goats were fed 10(5) Toxoplasma gondii oocysts and were monitored twice weekly for 8 wk for rectal temperature, clinical signs, parasitemia, and antibody levels by indirect fluorescence antibody test (IFAT), latex agglutination test (LAT), and indirect enzyme-linked immunosorbent assay (ELISA). After 8 wk, all goats were killed, and samples of heart, skeletal muscle, brain, lymph nodes, kidneys, and liver were bioassayed in mice. Anorexia, fever, and lethargy were observed from day 3 to day 7 postinfection (PI). Parasitemia was detected by bioassay in 50% of infected goats from day 7 to day 14 PI. Viable T. gondii organisms were isolated from all infected goats. Antibodies to T. gondii were detected in some animals on day 10 PI by IFAT and LAT and on day 14 PI by ELISA. The infected goats were seropositive on day 17 PI.

Seroprevalence of Neospora caninum in dairy cattle in Bahia, Brazil.

Sera collected from 447 dairy cattle on 14 dairy farms were tested for Neospora caninum antibodies by use of an immunofluorescent antibody technique. Positive reactions with titres > or =1:200 were found in 63 (14.09%) of animals. Neospora positive sera were also tested for Toxoplasma gondii antibodies by using a commercial latex agglutination test. Antibodies to T. gondii were detected in 3 (4.76%) of 63 N. caninum positive sera. These results indicate that N. caninum infection is widespread among dairy cattle in Bahia state.

Serological survey of antibodies to Toxoplasma gondii in goats, sheep, cattle and water buffaloes in Bahia State, Brazil.

Serum samples from 439 goats, 240 sheep, 194 cattle and 104 water buffaloes were tested for antibodies to Toxoplasma gondii by a latex agglutination test. Antibodies to T. gondii were found in 28.93% of goats, 18.75% of sheep, 1.03% of cattle and 3.85% of water buffaloes, at a dilution of 1:64. The highest titres observed in goats, sheep, cattle and water buffaloes were 1:2048, 1:2048, 1:64 and 1:512, respectively.

Ovine toxoplasmosis: seroconversion during pregnancy and lamb birth rate in Uruguayan sheep flocks.

Specific anti-Toxoplasma gondii antibodies were analyzed by latex agglutination during the 5 months of pregnancy in the serum of sheep from two flocks in the Uruguayan north-west: one flock (79 sheep) kept under an intensive management system and the other (494 sheep) kept under an extensive management system. Titers obtained using the latex test correlated with those obtained by indirect immunofluorescence. Pregnancy was confirmed by laparoscopy in all sheep from the smaller flock and by determination of serum progesterone levels in the seroconverted sheep from the larger flock. The percentage of sheep originally exhibiting significant serum levels of anti-T. gondii antibodies (13.9% in the smaller flock and 28.5% in the larger one) as well as the observed levels of seroconversion (22.8% and 7.7%, respectively), indicated a high prevalence of this infection in the north-west of Uruguay. In addition, birth rates of seroconverted sheep in the larger flock were significantly different from those of non-seroconverted animals. These results, although preliminary, suggest that Toxoplasma infection would produce considerable economic losses in extensive ovine production in Uruguay.