A fluorescence polarization assay using recombinant protein ESAT-6 for the detection of antibodies against pathogenic Mycobacterium bovis in bovine.

Background: Bovine tuberculosis (bTB) is a major bacterial disease that causes significant economic disruption across the globe. Aims: Our study was based on using a fluorescence polarization assay (FPA) that used fluorescein-labeled ESAT-6 protein to detect Mycobacterium bovis antibodies in bovine serum. Methods: The ESAT-6 protein was used in a FPA. Positive TB reactors were determined by the comparative intradermal test (CID) and interferon gamma test (IFN-γ). Antibodies against M. bovis were detected using a fluorescein isothiocyanate (FITC) labeled tracer and a whole culture FITC labeled tracer in the positive cattle. Results: Of the 192 animals tested for bTB, 37 were found to be positive by either the CID or IFN-γ assays. Using the mP values from five culture-positive serum samples, a cutoff value of more than >127 mp provided the best discrimination between positive reactors and negative bTB animals. The ESAT-6 results of FPA in comparison with CID results revealed sensitivity of 92.9% and specificity of 64.6%, and in comparison with results IFN-γ, showed sensitivity of 95.7% and specificity of 49%. FPA using FITC labelled ESAT-6 as a tracer has better sensitivity (95.7%) and specificity (49.1%) than IFN-γ test in humoral immune response in animals. Conclusion: This work revealed that the ESAT-6 protein as an antigen can be used in diagnosing bTB using a practical and sensitive humoral test.

Patho-epidemiological and risk factor studies for detection of Neospora-associated abortion in cattle and buffaloes in Punjab, India.

The diagnosis of abortion in livestock is difficult. In this paper, Neospora caninumassociated abortion in cattle and buffaloes in India is reported for the first time. A total of 184 animals (cattle, n = 133; water buffaloes, n = 51) were randomly selected for seroprevalence studies from 28 farms with a history of abortion. Antibodies to N. caninum were detected using a commercial competitive enzymelinked immunosorbent assay (c-ELISA); the percentage seropositivity was 10.5% for cattle and 21.6% for buffaloes. Risk factors such as species, i.e. buffaloes in comparison to cattle (odds ratio [OR] = 2.33, calculated probability [p] = 0.05), disposal of foetus and foetal membranes by throwing them into the field, in comparison with burning/burial (OR = 2.63, p = 0.03), and origin of the animal, i.e. purchased from outside in comparison to born on the farm (OR = 4.69, p = 0.002), were significantly associated with N. caninum seropositivity in univariate logistic regression. In multivariate analysis, only two risk factors, animal purchased from outside (OR = 6.15, p = 0.001) and buffaloes (OR = 3.20, p = 0.01), were significantly associated with Neospora seropositivity at a p-value < 0.05. Histopathological examination of aborted foetal tissues (cattle, n = 13; buffaloes, n = 8) revealed N. caninum tachyzoites in the heart and liver of three foetuses, and the diagnosis was confirmed by positive immunoreactivity to anti-N. caninum polyclonal antibody in the placenta of one foetus. To the authors' knowledge, this is the first report of confirmed neosporosis abortion in cattle and buffaloes in India.

Les causes d'avortement chez le bétail sont difficiles à déterminer. Cet article constitue le premier rapport faisant état d'une association entre des avortements survenus chez des bovins et des buffles en Inde et le parasite Neospora caninum. Afin de déterminer la prévalence d'anticorps anti-N. caninum dans 28 élevages ayant présenté des cas d'avortement, 184 animaux provenant de ces élevages (bovins, n = 133 ; buffles domestiques, n = 51) ont été sélectionnés de manière aléatoire et soumis à une analyse sérologique. Une épreuve immuno-enzymatique commerciale de compétition (cELISA) a été utilisée pour la détection d'anticorps vis-à-vis de N. caninum ; la prévalence sérologique était de 10,5 % chez les bovins et de 21,6 % chez les buffles. Une analyse univariée appliquant un modèle de régression logistique a permis de déterminer les facteurs de risque suivants comme étant associés à la présence d'anticorps vis-à-vis de N. caninum : l'espèce (buffle vs bovin) (rapport de cotes ou odds ratio [OR] = 2,33, probabilité calculée [p] = 0,05), le mode d'élimination du foetus et des membranes foetales (rejetées au sol vs incinérées ou enfouies) (OR = 2,63, p = 0,03), et l'origine de l'animal (acheté à l'extérieur vs né dans l'élevage) (OR = 4,69, p = 0,002). L'analyse multivariée a fait apparaître une corrélation significative (valeur de p < 0,05) entre la présence d'anticorps vis-à-vis de Neospora et deux facteurs de risque seulement, à savoir l'achat de l'animal à l'extérieur (OR = 6,15, p = 0,001) et l'espèce bubaline (OR = 3,20, p = 0,01). L'examen histopathologique des tissus d'avortons (bovins, n = 13 ; buffles, n = 8) a révélé la présence de N. caninum sous forme de tachyzoïtes dans le coeur et le foie de trois foetus ; une réaction immunitaire déclenchée par des anticorps polyclonaux anti-N. caninum dans le placenta de l'un des foetus a confirmé ce diagnostic. À la connaissance des auteurs, il s'agit de la première observation rapportée et confirmée en Inde d'avortements dus à la néosporose chez des bovins et des buffles.

Diagnosticar las causas de un aborto en el ganado vacuno no es tarea fácil. Los autores describen, por primera vez en la India, casos de aborto ligados a Neospora caninum en ganado vacuno y búfalos. Tras seleccionar aleatoriamente un total de 184 animales (vacunos, n = 133; búfalos de agua, n = 51) en 28 explotaciones que habían sufrido casos de aborto, se efectuaron estudios de prevalencia sérica. Para detectar los anticuerpos contra N. caninum se empleó un ensayo inmunoenzimático (ELISA) de competición disponible en el mercado, que arrojó un porcentaje de seropositividad de un 10,5% en el caso del ganado y de un 21,6% en los búfalos. Analizando los resultados por regresión logística de una sola variable, se observó que la seropositividad para N. caninum presentaba una correlación significativa con factores de riesgo como: la especie, esto es, búfalos en comparación con vacunos (razón de probabilidades [OR] = 2,33, probabilidad calculada [p] = 0,05); la eliminación del feto y las membranas fetales arrojándolas al campo, en comparación con su incineración o inhumación (OR = 2,63, p = 0,03); y la procedencia del animal, esto es, los animales adquiridos del exterior respecto de los nacidos en la explotación (OR = 4,69, p = 0,002). Al efectuar un análisis multifactorial solo se encontraron dos factores de riesgo significativamente relacionados con la seropositividad para Neospora a un valor de p < 0,05: la adquisición de animales del exterior (OR = 6,15, p = 0,001); y el hecho de que fueran búfalos (OR = 3,20, p = 0,01). El estudio histopatológico de tejidos de fetos abortivos (vacunos, n = 13; búfalos, n = 8) reveló la presencia de taquizoítos de N. caninum en el corazón y el hígado de tres fetos, diagnóstico confirmado por la inmunorreactividad positiva frente a anticuerpos policlonales contra N. caninum en la placenta de un feto. Hasta donde saben los autores, se trata de los primeros casos de aborto por neosporosis en ganado vacuno y búfalos que se describen y confirman en la India.

ISMap02 element targeted nested polymerase chain in the detection of Mycobacterium avium subsp. paratuberculosis in fecal samples of cattle and buffaloes.

BACKGROUND AND AIM: Johne's disease is chronic granulomatous enteritis which affects ruminants. There are many diagnostic approaches for the detection of Mycobacterium avium subsp. paratuberculosis (MAP) of which molecular detection methods using various elements are less time consuming and more accurate. The present study was conducted using ISMap02 element for nested polymerase chain reaction (nPCR) based detection of MAP in fecal samples. The aim was to test the sensitivity and specificity of the ISMap02 element and also to use this element for the detection of MAP in fecal samples of cattle and buffaloes. MATERIALS AND METHODS: A total of 211 fecal samples of cattle and buffaloes from different herds around Ludhiana aged between 2 and 13 years were collected, and DNA extraction was done from these samples. The nPCR was carried out for the detection of MAP in fecal samples. RESULTS: The ISMap02 element was specific for the detection of MAP only and showed a sensitivity of detection of 7.6 fg/µL of the standard genomic DNA. Among the 211 fecal samples of cattle and buffaloes tested for the ISMap02 element, 18 samples (8.5%) were positive for MAP. CONCLUSION: The ISMap02 element is specific and sensitive for the detection of MAP in various samples, and when used in nPCR format, it can increase the sensitivity of detection.

Comparison of diagnostic tests for the detection of bovine brucellosis in the natural cases of abortion.

Rapid and precise diagnosis in natural field cases of bovine abortion caused by Brucella abortus warrants the use of the most sensitive and reliable diagnostic methods. In the present study, bacterial isolation, serology, gross, histopathology, immunohistochemistry and polymerase chain reaction technique(s) were applied. Sero-prevalence studies showed the rate of 28.86% positive cases using the competitive ELISA. Histopathological changes were mainly seen in the placenta, fetal lungs, kidney, liver and spleen. Immunohistochemical (IHC) staining of Brucella spp. was evident as brown, finely granular intracytoplasmic staining in trophoblasts of placental sections and in section(s) of liver, lung, kidney and spleen. Twenty-eight out of the 103 samples (17 from stomach contents, 3 from placental cotyledons, 2 from vaginal discharges and 6 from pooled fetal tissues) produced 193 bp amplicon specific for Brucella genus. Moreover, the species-specific primers amplified a 498 bp amplicon which corresponded to B. abortus. Comparison of diagnostic tests revealed PCR and IHC provide a reliable test for the diagnosis of bovine brucellosis in aborted fetal tissue and placental cotyledons whereas serology is most important for detection of Brucella positive animals in a herd.

Epidemiological and immunopathological studies on Porcine parvovirus infection in Punjab.

AIM: The aim of this study was to get the first-hand knowledge about the seroprevalence of Porcine parvovirus (PPV) in Punjab and a diagnosis of PPV from abortion cases of swine using gross, histopathological, and immunohistopathological techniques to observe the tissue tropism of the virus strain. MATERIALS AND METHODS: Tissue samples from the reproductive tract of pig (n=32), placental tissue (n=10), and aborted fetuses (n=18) were collected from Postmortem Hall of the Department of Veterinary Pathology, GADVASU, field outbreaks and from butcher houses in and around Ludhiana. These samples were processed for histopathological and immunohistochemical (IHC) studies. For seroprevalence study, 90 serum samples of different sex and age were collected from 15 swine farms of Punjab and were subjected to indirect enzyme linked immunosorbent assay using commercial kit. RESULTS: Overall, seroprevalence of PPV was found to be 41.1%. Sex and age related difference in the prevalence was noted. In abortion cases grossly congested and emphysematous lungs, congested internal organs with fluid in abdominal cavity and congestion in brain, changes were noted in fetuses, while diffuse hemorrhages and edema was observed in placental tissue. Histopathologically, the most frequent fetal lesions in aborted fetuses were noted in lungs, liver, and brain. IHC staining revealed PPV antigens in sections of heart, liver, lung, spleen, brain, lymph node of fetuses, placenta, and uterus of sow. Gross, histopathological, and IHC examination of the samples confirmed 5 fetus, 2 placenta and 3 female reproductive samples positive for parvovirus infection. CONCLUSIONS: Seroprevalence results may serve as a support either in prevention or control of the disease. IHC is the sensitive technique for diagnosis of PPV associated with the reproductive tract of swine and was found to supplement the gross and histopathological alterations, respectively, associated with the disease.

Evaluation of lateral flow assay as a field test for investigation of brucellosis outbreak in an organized buffalo farm: A pilot study.

AIM: The aim was to evaluate lateral flow assay (LFA) as a field test for investigation of brucellosis outbreak in organized buffalo farm. MATERIALS AND METHODS: A total of 153 serum samples were tested to detect the presence of brucella antibodies by LFA and three other serological tests i.e. rose bengal plate test (RBPT), protein G based indirect enzyme-linked immunoassay (iELISA), and competitive ELISA (cELISA). The performances of LFA and other serological tests were evaluated using OIE complaint cELISA as the gold standard. RESULTS: Serological tests revealed 50% of the animals were seropositive for Brucella antibodies and correlated with clinical history of abortions, infertility, and productive failures. The newly developed assay showed 87.1% and 92.6% sensitivity and specificity, which was even higher than the specificity of RBPT. CONCLUSIONS: The investigation proved the potential usefulness of LFA for field diagnosis of brucellosis in the regions where laboratory facilities are limited.

Comparison of diagnostic tests for diagnosis of infectious bovine rhinotracheitis in natural cases of bovine abortion.

Rapid and precise diagnosis plays a pivotal role in implementing suitable control measures in natural field cases of bovine abortion due to infection with bovine herpesvirus (BHV)-1. In the present study, serology, virus isolation, histopathology, immunohistochemistry (IHC) and real-time polymerase chain reaction (PCR) for amplification of the gene encoding glycoprotein B were applied for diagnosis of infectious bovine rhinotracheitis (IBR) in cases of abortion. The seroprevalence of IBR in the population studied was 26.3% as determined by indirect enzyme-linked immunosorbent assay. BHV-1 abortions occurred between 4 and 8 months of gestation with an average gestational age of 6 months. Affected placentae showed necrosis of chorionic villi and of the endothelium of small villous blood vessels with characteristic intranuclear (IN) acidophilic inclusion bodies. Similar inclusions were also seen in most of the tissues examined. BHV-1 antigen was identified immunohistochemically in necrotic foci in the liver, the endothelium of placental blood vessels, the bronchial epithelium and hepatocytes. Lesions in the brain also had IN inclusion bodies that labelled positively by IHC. Eighteen samples (nine of stomach content, two of placental cotyledons, five of pooled fetal tissue and two of vaginal discharge) out of 84 tested were positive by real-time PCR for BHV-1.

Molecular epidemiology of Mycobacterium avium subspecies paratuberculosis: IS900 PCR identification and IS1311 polymorphism analysis from ruminants in the Punjab region of India.

Johne's disease is chronic granulomatous infectious enteritis of animals caused by Mycobacterium avium subspecies paratuberculosis. A total of 153 animals from 19 dairy farms, 2 gaushalas (unproductive-animal rehabilitation centers), 2 goat and 2 sheep farms from different districts of the Punjab region were selected on the basis of clinical signs of disease. All samples from cattle (n=86), buffalo (n=34), goat (n=25) and sheep (n=26) were subjected to Ziehl-Neelsen staining and DNA extraction by a freeze and thaw method. Ziehl-Neelsen staining detected 71% samples positive for acid-fast bacilli whereas IS900 PCR detected 55% positive for Map DNA. IS1311 PCR-REA analysis of IS900 positive samples revealed 'Bison' type as the most prevalent (82%) genotype of Map, infecting all domestic ruminants. 'Cattle' type was present in a minority of cases (15%) from cattle, buffaloes and goats. This is the first report of 'Cattle' type Map from buffalo and goat species in India.

Molecular detection and typing of Mycobacterium avium subspecies paratuberculosis from milk samples of dairy animals.

Genotyping of Mycobacterium avium subspecies paratuberculosis (MAP) is important for precise classification of bacterium and for understanding the molecular epidemiology. The present study reports detection and typing of the MAP from milk. On the basis of clinical signs of diarrhea and/or weakness, the dairy animals suspected for Johne's disease were screened by Ziehl-Neelsen staining of fecal samples. The milk samples from 13 selected animals were processed for DNA extraction and direct IS900 polymerase chain reaction (PCR). MAP identified by IS900 PCR was genotyped using IS1311 PCR-restriction endonuclease analysis (REA). IS900 milk PCR revealed 30.8% animals positive for MAP, including 40% of the moderate and 50% of the heavy fecal shedders. All infected animals showed Bison type MAP in IS1311 PCR-REA. IS900 PCR can be used for screening of milk for MAP; however, the method needs to be evaluated for subclinical cases. IS1311 PCR-REA results indicated the predominance of Bison type MAP in the dairy animals of this region.

[In vitro evaluation of mercury leakage from dental amalgam using atomic absorption spectrophotometry]. / Valutazione in vitro del rilascio di mercurio dall'amalgama dentale mediante spettrofotometro ad assorbimento atomico.

OBJECTIVE: The use of silver amalgam as a tooth filler is under constant critical review because of its mercury content. After a review of the literature on this subject, in vitro spectrophotometry was used to assay the release of mercury by these amalgams in basal conditions. METHODS: The experiment was conducted in two phases using standard doses of amalgam. In Phase 1 Black Class I cavities were created in extracted teeth that were big enough to take the required dose of amalgam. In some cases the material was inserted in a single operation, while in others the amalgam was introduced in three stages. Some of the cavities were cleansed with cotton buds bathed in ethyl alcohol, while others were not. All the drilled teeth were imbued in a bath of artificial saliva held at a constant temperature. In the second experiment, crushed amalgam was immersed in a similar bath of artificial saliva but otherwise untreated. The saliva was assayed after different time lapses using atomic absorption spectrophotometry and the FIAS technique. RESULTS: The results showed extremely variable but always modest quantities of free mercury in the artificial saliva. CONCLUSIONS: The findings suggest that not all the mercury available in the amalgamation phase is involved in the formation of the crystalline reticulum and that the percentage of mercury bonded is different every time. While the titre of free mercury encountered was always extremely low and hard to predict, it cannot be ignored.