Number Of Positive Samples Research Articles

Real-time PCR quantification of 9 periodontal pathogens in saliva samples from periodontally healthy Korean young adults.

PurposeFew studies have examined periodontal pathogens from saliva samples in periodontally healthy young adults. The purposes of this study were to determine the prevalence of periodontopathic bacteria and to quantify periodontal pathogens in saliva samples using real-time polymerase chain reaction (PCR) assays in periodontally healthy Korean young adults under 35 years of age.MethodsNine major periodontal pathogens were analyzed by real-time PCR in saliva from 94 periodontally healthy young adults. Quantification of Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Tannerella forsythia, Treponema denticola, Prevotella intermedia, Fusobacterium nucleatum, Campylobacter rectus, Peptostreptococcus anaerobius, and Eikenella corrodens was performed by DNA copy number measurement.ResultsF. nucleatum and E. corrodens were detected in all subjects; the numbers of positive samples were 87 (92.6%), 91 (96.8%), and 90 (95.7%) for P. gingivalis, P. anaerobius, and C. rectus, respectively. Other pathogens were also detected in periodontally healthy subjects. Analysis of DNA copy numbers revealed that the most abundant periodontal pathogen was F. nucleatum, which was significantly more prevalent than all other bacteria (P<0.001), followed by P. anaerobius, P. gingivalis, E. corrodens, C. rectus, and T. denticola. There was no significant difference in the prevalence of each bacterium between men and women. The DNA copy number of total bacteria was significantly higher in men than in women.ConclusionsMajor periodontal pathogens were prevalent in the saliva of periodontally healthy Korean young adults. Therefore, we suggest that the development of periodontal disease should not be overlooked in periodontally healthy young people, as it can arise due to periodontal pathogen imbalance and host susceptibility.

Role of Immunohistochemistry in the Diagnosis of Leptospirosis in Neotropical Primates

Background: Leptospirosis is considered the most widespread zoonosis worldwide, occurring more frequently in tropical and developing regions. The aim of the present study was to detect the presence of Leptospira spp. in different primate tissues, using immunohistochemical (IHC) assays, taking advantage of the considerable number of necropsies compatible with a diagnosis of leptospirosis in neotropical primates at the Animal Pathology Laboratory (APL) of the University of Passo Fundo (UPF) in the northern region of Rio Grande do Sul.Materials, Methods &amp; Results: Paraffin-embedded primate tissue samples were selected from necropsy examinations and subjected to IHC. The streptavidin-biotin-peroxidase method was used with diaminobenzidine chromogen (DAB) to verify immunostaining. Of the101 primates tested for Leptospira spp., 51.48% were positive; taining was distributed between lung (76.92%), liver (44.23%), and kidney (32.69%) tissue. Analysis of the combined anatomopathological verification data of the studied organs revealed a high frequency of lesions commonly observed in the tissues of animals exposed to the pathogen. For complementary diagnosis, an anti-Leptospira spp. antibody test was performed in primates at the UPF-Zoo, from which a population of the necropsied animals originated. The microscopic agglutination test (MAT) was utilized, which demonstrated 90.47% positivity in 21 individuals; sejroe and panama were the most frequent serovars.Discussion: Different intensities of tissue immunostaining were observed. Areas of fragmented or diffuse staining were considered to indicate equal positivity to that indicated by areas of staining with preserved morphology. Of 52 Leptospirapositive primates, most presented some degree of staining in lung samples, which shows a high level of involvement for this organ in primate leptospirosis. Conventional pathological diagnostic methods do not allow fort issue antigen recognition, thus making the IHC technique important to facilitate conclusive antigen sample verification. In the liver, leptospires were detected mostly between the sinusoids, hepatocytes, and Kupffer cells. In kidney tissues, staining indicated small agglomerates in the tubular lumen, interstitium, and glomeruli. All these forms of presentation have been previously reported. Considering that we detected the highest number of positive samples in lung tissue, followed by those from liver and kidney tissue, we argue that the IHC technique, when applied to samples of these three tissues, decreases the chance of false negatives. Anatomopathological studies of primate leptospirosis are scarce. In dogs, renal lesions are characterized by the necrosis and degeneration of tubular epithelium, cellular debris, and hyaline cylinders. In the liver, hepatocyte cord dissociation and biliary pigment accumulation within the canaliculi and hepatocellular necrosis are observed. These findings are similar to those from our study. In the lung, diffuse alveolar lesions are reported, with hemorrhage and edema, in addition to capillaritis. The high frequency of Leptospira-positive animals determined by serological examination was consistent with the IHC findings, thus confirming the pathogen’s high prevalence in neotropical primate populations in the studied region. Serological surveys on primate populations have already been carried out and have revealed frequency and serovar variations between regions. Immunohistochemical examination allows the detection of leptospires in various tissues and should be used based on the characteristics of the investigated case.

Can pruning help maintain vitality of ash trees affected by ash dieback in urban landscapes?

Hymenoscyphus fraxineus causes a destructive invasive tree disease known as ash dieback threatening the survival of common ash not only in the forests, but also in urban and landscape settings. Pruning is a potential management practice that could help maintain tree vitality and aesthetics in parks, gardens, alleys and recreation areas, as well as maintaining veteran trees having high heritage or cultural value, or trees with high genetic importance (e.g. located in clonal seed orchards). In this study we investigated the maximum distance proximal to the lesion margin at which H. fraxineus can be detected on individual branches infected by the fungus in order to provide recommendations for pruning. Pruning of branches was carried out on 38 trees in southern Sweden. Tissue samples including bark and wood were collected from the margin of the lesion and at 5cm intervals proximal to the lesion. Molecular analysis revealed presence of H. fraxineus in 91.3% of the investigated lesions. The proportion of lesions at which H. fraxineus could be detected declined with increasing distance from the lesion margin, with a significant reduction in the number of positive samples at 10cm proximal to the margin. At 30cm from the lesion edge the pathogen was never detected. Our results suggest that routine pruning may help maintain the vitality of younger trees. Pruning branches at least 35cm from visible, active lesions in the bark should exclude the fungus and therefore reduce the probability of stem infection by H. fraxineus, however this cultural control tactic may only be economically feasible for high value amenity trees.

CTL-DNNet: Effective Circular Traffic Light Recognition with a Deep Neural Network

This paper proposed an algorithm for a deep learning network for identifying circular traffic lights (CTL-DNNet). The sample labeling process uses translation to increase the number of positive samples, and the similarity is calculated to reduce the number of negative samples, thereby reducing overfitting. We use a dataset of approximately 370[Formula: see text]000 samples, with approximately 20[Formula: see text]000 positive samples and approximately 350[Formula: see text]000 negative samples. The datasets are generated from images taken at the Beijing Garden Expo. To obtain a very robust method for the detection of traffic lights, we use different layers, different cost functions and different activation functions of the depth neural network for training and comparison. Our algorithm has evaluated autonomous vehicles in varying illumination and gets the result with high accuracy and robustness. The experimental results show that CTL-DNNet is effective at recognizing road traffic lights in the Beijing Garden Expo area.

PREVALENCE OF PARASITES WITH ZOONOTIC POTENTIAL IN SOIL FROM THE MAIN PUBLIC PARKS AND SQUARES IN CAXIAS DO SUL, RS, BRAZIL

Soil contamination in parks and public squares caused by animal feces has been one of the main forms of parasitic contamination, becoming therefore a public health problem. The purpose of this study was to determine the prevalence of parasites in the soil in major parks and public squares in Caxias do Sul, RS, Brazil. Two samplings were performed at each location, the firstin summer (February) and the second in winter (July) in 2015. Soil samples were collected in five spots in ten parks/public squares, per season and examined using the Hoffman, Pons &amp; Janer (HPJ), Faust and Rugai, Mattos and Brisola methods. Among the 100 samples analyzed by HPJ and Rugai, Mattos and Brisola, 64% and 58% respectively, proved positive in summerand 28% and 60% in winter. The Faust method showed the same number of positive samples in both seasons (12% each). The following parasites were found: Cystosospora sp oocysts (4%), Entamoeba coli cysts (1%), Strongyloides stercoralis suggestive larvae (0.3%), Endolimax nanacysts (1%) and Ancylostoma sp suggestive larvae (33.3%) with the highest prevalence. Both parks and squares presented parasites with zoonotic potential. Thus, it is necessary to highlight the importance of educational measures regarding the use of these locations as well as the cleaning of public areas with the removal of animal excrements.

Salmonella serovars and their distribution in Nigerian commercial chicken layer farms.

Commercial poultry farms (n° 523), located in all the six regions of Nigeria were sampled with a view to generate baseline information about the distribution of Salmonella serovars in this country. Five different matrices (litter, dust, faeces, feed and water) were collected from each visited farm. Salmonella was isolated from at least one of the five matrices in 228 farms, with a farm prevalence of 43.6% (CI95[39.7–48.3%]). Altogether, 370 of 2615 samples collected (14.1%, CI95[12.8; 15.5%]) contained Salmonella. Considering the number of positive farms and the number of positive samples, it was evident that for the majority of the sampled farms, few samples were positive for Salmonella. With regard to the matrices, there was no difference in Salmonella prevalence among the five matrices considered. Of the 370 isolates serotyped, eighty-two different serotypes were identified and Salmonella Kentucky was identified as having the highest isolation rate in all the matrices sampled (16.2%), followed by S. Poona and S. Elisabethville. S. Kentucky was distributed across the country, whereas the other less frequent serovars had a more circumscribed diffusion. This is one of few comprehensive studies on the occurrence and distribution of Salmonella in commercial chicken layer farms from all the six regions of Nigeria. The relatively high prevalence rate documented in this study may be attributed to the generally poor infrastructure and low biosecurity measures in controlling stray animals, rodents and humans. Data collected could be valuable for instituting effective intervention strategies for Salmonella control in Nigeria and also in other developing countries with a similar poultry industry structure, with the final aim of reducing Salmonella spread in animals and ultimately in humans.

Epidemiological analyses on African swine fever in the Baltic countries and Poland.

African swine fever virus (ASFV) has been notified in the Baltic countries and the eastern part of Poland from the beginning of 2014 up to now. In collaboration with the ASF‐affected Member States (MS), EFSA is updating the epidemiological analysis of ASF in the European Union which was carried out in 2015. For this purpose, the latest epidemiological and laboratory data were analysed in order to identify the spatial–temporal pattern of the epidemic and a risk factors facilitating its spread. Currently, the ASF outbreaks in wild boar in the Baltic countries and Poland can be defined as a small‐scale epidemic with a slow average spatial spread in wild boar subpopulations (approximately from 1 in Lithuania and Poland to 2 km/month in Estonia and Latvia). The number of positive samples in hunted wild boar peaks in winter which can be explained by human activity patterns (significant hunting activity over winter). The number of positive samples in wild boar found dead peaks in summer. This could be related to the epidemiology of the disease and/or the biology of wild boar; however, this needs further investigation. Virus prevalence in hunted wild boar is very low (0.04–3%), without any apparent trend over time. Apparent virus prevalence at country level in wild boar found dead in affected countries ranges from 60% to 86%, with the exception of Poland, where values between 0.5% and 1.42%, were observed. Since the beginning of the epidemic, the apparent antibody prevalence in hunted wild boar has always been lower than the apparent virus prevalence, indicating an unchanged epidemiological/immunological situation. The risk factor analysis shows an association between the number of settlements, human and domestic pigs population size or wild boar population density and the presence of ASF in wild boar for Estonia, Latvia and Lithuania.

Serological Diagnostic Assays for Detection of Ns1 Antigen, IGM and IGG Antbodies to Dengue Virus

Complications in managing the dengue virus infections include the lack of rapid diagnostic procedures and at the same time the symptoms of dengue infection are often confused with those of other diseases. Two commercial rapid serological diagnostic kit methods (Dengue Day 1 test, J Mitra and Co. Pvt. Ltd., New Delhi, ImmunoComb II Dengue IgM/IgGBispot kit (Orgenics Pvt. Ltd., Israel)were evaluated for the detection of NS1 antigen, Immunoglobulin IgG and IgM specific to dengue virus in the serum samples of patients suffered with dengue acute primary infection and secondary infection. The total assay time was 20 min-2hrs. The results of these methods were compared with the gold standard assay methods Dengue IgM-Capture Microplate ELISA and Dengue Indirect IgG ELISA (Pan Bio, Brisbane, Australia). The total assay time was 6-7hrs. Nine serum samples were positive to NS1 antigen and negative to IgG and IgM by Dengue day 1 test. The results of Bispot assay method revealed that, the number of IgG positive samples was 11, IgM positive samples were 31 and both IgG and IgM positive samples were 8. Majority of the positive cases were noticed in the age group 35-68 years and males were more prone to dengue infection while comparing with females. By performing IgM MICROLISA, 34 samples were positive which in turn indicated that, three of them were false negative by the immune comb bispot method giving a sensitivity of 91.17%. Through indirect IgG ELISA, the number of positive samples was 15 and four of the 15 positive samples of IgG were false negative by the immuo comb bispot method giving a sensitivity of 73.33%. The gold standard ELISA methods were more efficient than rapid serological tests and gave an overall sensitivity of 99%. Thus the alternative of an assay that is to be used in the diagnosis of dengue infections depends on factors like laboratory infrastructure, preference and availability of equipment. The allied performance of the Rapid test, followed by confirmation with MAC-ELISA on those samples, ensures both speediness as well as quality of reported results.

Evaluation of Three Rapid Diagnostic Test Kits in the Diagnosis of &amp;lt;i&amp;gt;Plasmodium&amp;lt;/i&amp;gt; Species Infections among Primary School Children in Baringo County, Kenya

Background: Due to the difficulty in implementing microscopy-based definitive diagnosis of malaria especially in resource-poor areas, rapid diagnostic test (RDT) kits as alternatives have been developed by different companies. Kits from different companies may differ in sensitivity and specificity. It is therefore imperative to conduct field evaluation of kits from different manufacturers before they are recommended for widespread use in diagnosis of malaria. The present study sought to evaluate the performance of malaria RDT kits from two manufacturers against microscopy in detection of Plasmodium spp. infections among primary school children in Baringo County, Kenya. Methods: Two cross-sectional comparative surveys to assess three RDT kits (CareStart HRP-2, SD Bioline Ag-Pf and SD Bioline Ag-Pf/Pan) were conducted on 561 pupils from three primary schools within the Kerio Valley of Baringo County, Kenya. Results: A total of 561 blood samples were tested for Plasmodium spp. within a period of four months. SD Bioline Ag-Pf/ Pan RDT kit had a higher sensitivity (90%) compared to that of CareStart HRP-2 (70%) but this was not statistically significant (McNemar’s χ2 = 0.5, df = 1, p = 0.4795). The sensitivity of SD Bioline Ag-Pf was the same as that of Care Start HRP-2 (82.4%). The number of positive samples by microscopy was relatively lower than those detected by RDT kits. Two cases of non-P. falciparum infections were detected by RDT kits during the second survey. Conclusion: The results of the present study suggest that the performance of the three kits in the diagnosis of P. falciparum infections was better than microscopy. The sensitivity of SD Bioline Ag-Pf/Pan was slightly greater than that of Care Start HRP-2, although not statistically different.

Effect of Pranayama on Ppar-γ, Nf-κB Expressions and Red Complex Microorganisms in Patients with Chronic Periodontitis – A Clinical Trial

Sudarshankriya pranayama is the control of breathing movements to regulate the energy flow throughout the body, which has shown to positively affect immune function, autonomic nervous system and psychologic-neuro pathways. The practice of pranayama has been proven to show several benefits such as reduction in stress levels, relieves anxiety and depression, increase in antioxidant levels, reduces insulin resistance and improves respiratory function. The objective of the study was to evaluate the levels of Peroxisome Proliferator-Activated Receptor Gamma (PPAR-γ), Nuclear Factor-Kappa B (NF-κB) and the presence of Red Complex Microorganisms (RCM) such as Treponema denticola, Porphyromonas gingivalis and Tannerella forsythia in the subgingivalpl aque samples of chronic periodontitis subjects before and after intervention with pranayama as an adjunct to Scaling and Root Planing (SRP). A total of 30 subjects (control group) were treated with SRP and 30 subjects (pranayama group) underwent SRP and pranayama for three months. Probing Pocket Depth (PPD), Clinical Attachment Level (CAL), Bleeding Index (BI) and Plaque Index (PI) were recorded and the presence of PPAR-γ, NF-κB and RCM were assessed at baseline and after three months using polymerase chain reaction. ANCOVA test was done to compare the clinical parameters between the groups. Fisher's Exact test was done to identify RCM and Mann-Whitney and Wilcoxon-signed test was used to identify the expression of NF-κB and PPAR-γ in the plaque samples. The change in the mean CAL from baseline to third month was significantly higher in pranayama group compared to control group (p≤0.05). There was a statistically significant reduction in the expression of NF-κB and increase in PPAR-γ expression levels in pranayama group on comparison with the control group (p<0.001). The reduction in number of positive samples with T.denticola, P.gingivalis and T.forsythia at third month post-intervention did not affect the change in the expression levels of NF-κB and PPAR-γ. The CAL showed significant improvement with reduction in the RCM, NF-κB and increase in PPAR-γ levels in subjects who underwent pranayama as an adjunct to SRP. In future, pranayama can be used as an additional treatment modality to provide a new dimension in treatment of periodontitis.

Microbiological safety and quality control of poultry products sold in retail chains of Saint Petersburg and Leningrad region.

Food safety and food raw materials is an important task which needs to be under government’s and public associations’ control, under enterprises which produce and sale them, and other economy departments. Nutrition is one of the most important factors influencing human health. To 70% alien substances of chemical and microbiological origin get into the human body with food. The heavy metals, nitrates, nitrites, pesticides, nitrosescomounds, mycotoxins, antibiotics, radionuclides and others are xenobiotics of chemical origin. The poultry products represented in commercial networks by poultry plants located in Saint-Petersburg and Leningrad region: “Roskar”, “Sinyavinskaya”, “North”, “Lebyazhye” were picked up for analysis. We used the bacteriological, virological, immunological, mycological, and statistical methods of research. Sanitary -hygienic or microbiological safety of food products was evaluated for the contenting of disease-causing (pathogenic) microorganisms such as bacteria groups, coliforms, Staphylococcus, Salmonella and etc. in them. The poultry products sold in commercial networks of St. Petersburg were picked up for analysis. After processing the statistical were revealed 6 nosological forms where the leading position has finding colibacillosis – 27%. The number of positive samples for streptococcosis – 18%, staphylococcus – 8%, aspergillosis–2.6%, salmonellosis – 1.8%, pasteurellosis–1.6 %. The microbiological control of food products which can be the sources of infections should be regular for providing inhabitants with harmless poultry products. It is necessary to adopt and to use the modern microbiological criteria of food safety that will allow to realize the monitoring of their conformity with sanitary norms. The results of these analysis shows that the quality of poultry products sold in commercial networks of Saint Petersburg and the Leningrad region meet the standards.

(628) - Is Routine Respiratory Viral PCR Testing in Lung Transplant Recipients Helpful?

Our unit adopted routine screening for a panel of respiratory viruses using quantitative PCR on all bronchoalveolar lavage (BAL) samples from LTx recipients since 2009. We evaluated diagnostic yield and usefulness of this approach. Retrospective examination of BAL viral PCR results in all LTx performed from January 2009 to December 2013 at our institution. Total number of samples and number of positive samples per patient were noted. Results were stratified by single (SLTx) vs. double (DLTx) lung recipients and by surveillance bronchoscopy (SB) vs. bronchoscopy clinically indicated by a drop in spirometry and/or radiographic changes (CB). 123 LTx were performed in the study period (35 SLTx, 88 DLTx; 70 males, mean age 52 yrs [range 17-65]; SLTx indications: IPF 25, other fibrosis 8, COPD 1, sarcoid 1; DLTx indications: CF 13, non-CF bronchiectasis 4, COPD 49, alpha 1 antitrypsin deficiency 8, obliterative bronchiolitis 3, IPF 2, other fibrosis 3, iPAH 4, CTEPH 1, sarcoid 1). Mean follow up was SLTx: 1001 days (range 10-2392) and DLTx 1034 (0-2344) during which 84 and 222 BAL viral PCR panels were performed respectively. 6 positive PCR results in 5 SLTx and 24 in 19 DLTx were noted (P=NS) at a median of 56 (range 40-845) vs 201 (range 3-1232) days post op (P=NS). Rhinovirus was most prevalent (18 samples vs. parainfluenza 6, adenovirus 4, respiratory syncytial virus 3, metapneumovirus 3). Rhinovirus co-existed with adenovirus in 2 patients and with parainfluenza in 1 patient. 7/228 (3%) SB vs 23/78 (29%) CB samples were PCR positive (P<0.05). Apart from 2 RSV episodes treated with oral ribavarin, no targeted therapy was implemented. Our data suggest a limited diagnostic yield from a respiratory viral PCR panel in routine surveillance BAL samples post LTx. Most viruses identified are self-limiting with no proven effective therapy and the cost of expensive routine testing is therefore unjustified. In contrast, clinically indicated BAL is 10 times more likely to reveal positive viral DNA. Freedom from respiratory viruses appeared numerically, though not statistically, shorter in SLTx recipients but this is confounded by the small sample size. Native lung factors may be relevant and this merits further investigation.

Mastitis Mikotik Akibat Terinfeksi Candida spp dan Trichosporon spp pada Peternakan Sapi Perah di Bogor, Bandung, dan Jakarta (MYCOTIC MASTITIS CAUSED BY CANDIDA SPP AND TRICHOSPORON SPP ON DAIRY FARM IN BOGOR, BANDUNG, AND JAKARTA)

Mycotic mastitis is a disease affecting dairy cows and the causative agent is fungi of yeast, and alsomolds, but the disease is dominantly caused by yeast. In Indonesia reported of the disease is very rare.Generally, the mycotic mastitis is chronic causing great economic losses. This study aim was to show thatthere are still cases of mycotic mastitis in dairy cows in the area of Bandung, Bogor and Jakarta. Isolationand identification of fungi that caused mycotic mastitis were performed from the positive milk samplesteseted with California Mastitis Test (CMT). Of the 184 positive samples mastitis tested with CMT, 71samples mycotic mastitis were obtained, The 71 samples consisted of 13 molds and 84 yeasts based onthe isolates findings. Consecutive areas where commonly found mycotic mastitis were Bogor (50%);Bandung (38%); and Jakarta (27%). The number of positive samples in mycotic mastitis caused by moldinfection findings obtained less than yeasts. The yeast of Trichosporon spp found 34 isolates which washigher than Candida spp at 26 isolates. The existence of this yeast contamination was associated with thehygiene of the cage and the dairy cows. Control needs to be done to reduce the cases of mycotic mastitis.

Studying the Elimination Pattern of Caprine Arthritis Encephalitis Virus in the Milk of Infected Females

Background: Small ruminants can be infected by lentiviruses, such as Maedi-Visna Virus (MVV) and Caprine ArthritisEncephalitis Virus (CAEV). The main route of transmission is via ingestion of contaminated colostrum and milk although vertical transmission can occur. Recently, several studies for molecular detection of CAEV in milk, using conventional PCR and real-time PCR are being carried out. Considering the elimination of CAEV through the milk of infected animals and the importance of this virus in the goat production, the aim of this study was to evaluate the elimination pattern of CAEV in milk, evaluating the frequency and the concentration eliminated during the lactation.Materials, Methods &amp; Results: A cohort of four negative females for CAEV was inseminated with semen experimentally infected with CAEV-Cork strain. They were located in stalls at the Hospital of Ruminants from School of Veterinary Medicine and Animal Science from University of São Paulo, Brazil. Goats received coast-cross hay, pellet feeding, mineral salt and water ad libitum. All females were observed every day during pregnancy. After lambing, kids received warm bovine colostrum and bovine milk powder during two months. Forty milk samples were collected at five-day interval during two months. A mixture of five milliliters from each teat was obtained and cDNA extraction was performed using DNA Mini Kit. Initially, real-time PCR was performed using an endogenous control for research of the constitutive gene (12S) for goats. Using positive samples in the first reaction, another reaction was performed using specific primers for lentiviruses based on the gag gene (conserved in retroviruses). In order to compare the results, nested-PCR was performed. After realtime PCR, cDNA was detected in samples from one female, corresponding to the day of calving, 14th, 20th, 25th, 35th and 40th day postpartum (15%; 6/40). The absence of amplified cDNA in thirty days postpartum, as well as in the final twenty days of lactation, was observed. Sample corresponding to the 7th day postpartum was not obtained. The virus concentration throughout lactation grew up until forty days postpartum. After this period, there was no cDNA amplification. In Nested PCR, positive results were detected in samples corresponding to the day of calving, 15th days, 20th days and 30th days postpartum, only.Discussion: cDNA was detected in samples from one positive female, during forty days postpartum, but not on the 30th. On the other hand, amplified cDNA was observed on 30th day by nested-PCR. In this case, a false negative result was observed after real-time PCR, probably because sample corresponding to 30th days may not have been properly homogenized, so that the fraction used in real-time PCR was not representative. A higher number of positive samples were expected due to the higher sensitivity of the technique used. The low viral concentration in the milk due to high antibody titers, for example, leaded to a small number of cells containing the agent, reducing the possibility of detection. cDNA was not detected in any sample from three infected females. A possible false-positive serological reaction or the very low viral concentration in milk samples could explain the negative results, although some animals might be infected by a strain that could not be recognized by PCR.

Assessment of the degree of concordance between the results of histopathological examination and bacterial culture in the diagnosis of bovine tuberculosis in Mexico

Bovine tuberculosis is a complex disease that is difficult to diagnose, control and eradicate and negatively impacts many farms. The objective of this study was to assess the degree of concordance between the results of histopathological examination and bacterial culture in the diagnosis of bovine tuberculosis lesions obtained in the laboratories certified by the Secretaría de Agricultura, Ganadería, Desarrollo Rural, Pesca y Alimentación (Ministry of Agriculture, Animal Production, Rural Development, Fisheries and Feeding) at the national level between January 2009 and December 2012 in Mexico. Tissue samples (10,818) from regular slaughter cattle that did not have tuberculin tests but had lesions suspected of having been caused by tuberculosis were sent to 10 authorized laboratories. Using Cohen’s kappa to measure the reliability of the diagnosis, a general concordance was obtained between the histopathological examination and bacterial culture results with a kappa of 0.634 and a 95% (0.618 – 0.650) confidence interval (CI), which shows good concordance between the two techniques at the national level. The laboratory in Chihuahua had the highest kappa [k=0.784, 95% CI (0.754–0.814)] and the laboratory of the La Laguna Region in the state of Coahuila had a low value of global concordance [k= 0.334, 95% CI (0.257–0.412)]. The number of positive samples in bacterial culture was low in Tamaulipas (21 samples), Sonora (41 samples), and Yucatán (45 samples) because tuberculosis prevalence in those states is ≤ 0.04%. The number of positive samples in bacterial culture was low in Tamaulipas (21 samples), Sonora (41 samples), and Yucatán (45 samples) because tuberculosis prevalence in those states is ≤ 0.04%. The reason for the disagreement between the two tests among some laboratories includes factors such as a lack of economic resources, infrastructure or personnel training. Correct sampling procedures, storage facilities, and shipping and sample processes are important in optimizing the bacteriological and histopathological results and obtaining correct diagnoses and differential diagnoses of bovine tuberculosis.

Developing high throughput quantitative PCR assays for diagnosing Ikeda and other Theileria orientalis types common to New Zealand in bovine blood samples

AIMS: To develop rapid, quantitative PCR (qPCR) assays using high resolution melt (HRM) analysis and type-specific TaqMan assays for identifying the prevalent types of Theileria orientalis found in New Zealand cattle; and to evaluate their analytical and diagnostic characteristics compared with other assays for T. orientalis.METHODS: Nucleotide sequences aligned with T. orientalis Buffeli, Chitose and Ikeda types, obtained from DNA extracted from blood samples from infected cattle, were used to design HRM and type-specific probe-based qPCR assays. The three type-specific assays were also incorporated into a single-tube multiplex qPCR assay. These assays were validated using DNA extracted from blood samples from cattle in herds with or without clinical signs of T. orientalis infection, other veterinary laboratory samples, as well as plasmids containing T. orientalis type-specific sequences. Diagnostic specificity (DSp) and sensitivity (DSe) estimates for the qPCR assays were compared to blood smear piroplasm results, and other PCR assays for T. orientalis. Copy number estimates of Ikeda DNA in blood were determined from cattle exhibiting anaemia using the Ikeda-specific qPCR assay.RESULTS: The T. orientalis type-specific and the HRM qPCR assays displayed 100% analytical specificity. The Ikeda-specific qPCR assay exhibited linearity (R2 = 0.997) with an efficiency of 94.3%. Intra-assay CV were ≤0.08 and inter-assay CV were ≤0.095. For blood samples from cows with signs of infection with T. orientalis, the DSp and DSe of the multiplex probe qPCR assay were 93 and 96%, respectively compared with blood smears, and 97 and 100%, respectively compared with conventional PCR assays. For the Ikeda-specific qPCR assay, the number of positive samples (n=66) was slightly higher than a conventional PCR assay (n=64). The concentration of Ikeda genomes in blood samples from 41 dairy cows with signs of infection with T. orientalis ranged between 5.6 × 104 and 3.3 × 106 genomes per µL of blood.CONCLUSIONS: All qPCR assays had improved specificity and sensitivity over existing conventional PCR assays for diagnosis of T. orientalis Ikeda. The burden of Ikeda DNA in blood was demonstrated using an Ikeda-specific qPCR assay with titrated synthetic gene target.CLINICAL RELEVANCE: Adoption of high-throughput DNA extraction and qPCR reduced T. orientalis and Ikeda diagnosis times. The Ikeda-specific qPCR assay provides a specific diagnosis for Ikeda in animals with signs of infection with T. orientalis and can be used to monitor the parasite load of Ikeda in blood.

Assessment of the degree of concordance between the results of histopathological examination and bacterial culture in the diagnosis of bovine tuberculosis in Mexico

Bovine tuberculosis is a complex disease that is difficult to diagnose, control and eradicate and negatively impacts many farms. The objective of this study was to assess the degree of concordance between the results of histopathological examination and bacterial culture in the diagnosis of bovine tuberculosis lesions obtained in the laboratories certified by the Secretaria de Agricultura, Ganaderia, Desarrollo Rural, Pesca y Alimentacion (Ministry of Agriculture, Animal Production, Rural Development, Fisheries and Feeding) at the national level between January 2009 and December 2012 in Mexico. Tissue samples (10,818) from regular slaughter cattle that did not have tuberculin tests but had lesions suspected of having been caused by tuberculosis were sent to 10 authorized laboratories. Using Cohen’s kappa to measure the reliability of the diagnosis, a general concordance was obtained between the histopathological examination and bacterial culture results with a kappa of 0.634 and a 95% (0.618 – 0.650) confidence interval (CI), which shows good concordance between the two techniques at the national level. The laboratory in Chihuahua had the highest kappa [k=0.784, 95% CI (0.754–0.814)] and the laboratory of the La Laguna Region in the state of Coahuila had a low value of global concordance [k= 0.334, 95% CI (0.257–0.412)]. The number of positive samples in bacterial culture was low in Tamaulipas (21 samples), Sonora (41 samples), and Yucatan (45 samples) because tuberculosis prevalence in those states is ≤ 0.04%. The reason for the disagreement between the two tests among some laboratories includes factors such as a lack of economic resources, infrastructure or personnel training. Correct sampling procedures, storage facilities, and shipping and sample processes are important in optimizing the bacteriological and histopathological results and obtaining correct diagnoses and differential diagnoses of bovine tuberculosis.

Evaluación de un panel reducido de cepas de leptospiras para el diagnóstico en humanos por microaglutinación (MAT)

To evaluate if the use of the 19 Leptospira strains panel suggested by the International Leptospirosis Society of World Health Organization for microagglutination allows confirmation of more cases that the 12 strains panel used in Argentina. Cross-sectional observational study. We studied 441 serum samples corresponding to Argentinean patients with suspected leptospirosis derived during from July to December, 2009 and from January to October, 2013. The same number of positive samples was obtained using the MAT with the 19 or 12 strains. In six cases a serovar of the expanded collection was presumably infecting, but always coagglutinated with strains of the reduced panel. In Argentina, the diagnosis of leptospirosis by MAT could be made using the reduced 12 strains panel, obtaining the same result in case detection as using the 19 strains panel. Additional information provided by the use of all strains could be the presumably infecting serogroup.

Molecular diagnostics and genetic characteristics of influenza A/H1N1 virus circulating in the territory of the West Kazakhstan in 2012–2014

No: 1437 Presentation at ESCV 2015: Poster 1 Molecular diagnostics and genetic characteristics of influenza A/H1N1 virus circulating in the territory of the West Kazakhstan in 2012–2014 N.G. Klivleoeva1,∗, G.V. Lukmanova1, T.I. Glebova1, M.G. Shamenova1, N.T. Saktaganov1, K.V. Duysenova2 1 Institute of Microbiology and Virology of the Ministry of Education and Science of the Republic of Kazakhstan, Kazakhstan 2 Atyrau Regional Center of Sanitary – Epidemiological Expertise, Kazakhstan Background: The purpose of this study was to analyze the molecular characteristics of influenza A/H1N1virus circulating in the territory of the West Kazakhstan in 2012–2014. Methods: Samples of clinicalmaterials (nasopharyngeal swabs) were collected in polyclinics and infectious diseases hospitals of the Atyrau oblast (West Kazakhstan) from people with symptoms ofARVI andpneumonia in the2012–2014period. The sampleswere tested by RT-PCR considering the results in the real-time mode and using AmliSens reagent kit produced by the Central Research Institute of Epidemiology (Moscow). Nucleotide sequences of HA andNA geneswere analyzed using the BlAST software (http://blast. ncbi.nlm.nih.gov), which allowed to determine the percentage of identity with the influenza virus isolates and strains available in the GenBank. PCR diagnostics of 337 clinical samples showed that the genetic material of influenza viruses was detected in 118 swabs (35% of the total number of samples tested). Influenza A/H1 viral RNA was detected in 27 samples (23.0% of the total number of positive samples), A/H3 viral RNA in 13 samples (11%). RNA of simultaneously both subtypes of influenza A virus (H1N1+H3N2) was also detected in six samples (5.0%). Influenza B viruses were not detected. These data suggest the influenza A epidemic of the mixed etiology with a predominance of influenza A/H1N1 virus. Molecular genetic characterization of the six A/H1N1 strains, isolated in the Atyrau region in 2012 and 2013, showed that these viruses by the hemagglutinin gene are similar to the reference strain A/swine/Guangdong/L3/2009 (H1N1) by 98-99%. When studying the NA gene sequence, it was established that isolates exhibited 99% identity with A/swine/Changhua/1993/2000 (H1N1), A/swine/USA/1976-M/1931 (H1N1) and A/swine/Guangdong/L3/2009 (H1N1), and 98% homology with A/swine/Iowa/1973 (H1N1). Two strains (A/Atyrau/880/12 and A/Atyrau/901/13), in addition to this, showed 99% identity with the classical swine viruses A/swine/Jamesburg/1942 (H1N1), A/swine/1931 (H1N1) and A/swine/Iowa/1973 (H1N1). The study of mutational changes in NA and M genes and comparative analysis of amino acid sequences in NA genes in comparison with a pandemic strain A/California/04/2009 (H1N1)v of American line and A/England/195/2009 (H1N1) of Eurasian line indicated that the Kazakhstan strains have more than 70 differences from the reference ones. Phylogenetic analysis showed that closely related strains of influenza virus belonging to the same cluster with the virus A/swine/Guandong/L3/2009 (H1N1), isolated in 2009 from swine in China, are circulating in the territory of the West Kazakhstan; the changes in HA gene do not lead to the changes in the antigen properties. In this way, the molecular biological analysis showed that the population of A/H1N1 strains of theWest Kazakhstan is genetically homogeneous, and within two epidemic seasons of the circulation has not undergone significant changes. http://dx.doi.org/10.1016/j.jcv.2015.07.057

Prevalence and effects of mycotoxins on poultry health and performance, and recent development in mycotoxin counteracting strategies

Extensive research over the last couple of decades has made it obvious that mycotoxins are commonly prevalent in majority of feed ingredients. A worldwide mycotoxin survey in 2013 revealed 81% of around 3,000 grain and feed samples analyzed had at least 1 mycotoxin, which was higher than the 10-year average (from 2004 to 2013) of 76% in a total of 25,944 samples. The considerable increase in the number of positive samples in 2013 may be due to the improvements in detection methods and their sensitivity. The recently developed liquid chromatography coupled to (tandem) mass spectrometry allows the inclusion of a high number of analytes and is the most selective, sensitive, and accurate of all the mycotoxin analytical methods. Mycotoxins can affect the animals either individually or additively in the presence of more than 1 mycotoxin, and may affect various organs such as gastrointestinal tract, liver, and immune system, essentially resulting in reduced productivity of the birds and mortality in extreme cases. While the use of mycotoxin binding agents has been a commonly used counteracting strategy, considering the great diversity in the chemical structures of mycotoxins, it is very obvious that there is no single method that can be used to deactivate mycotoxins in feed. Therefore, different strategies have to be combined in order to specifically target individual mycotoxins without impacting the quality of feed. Enzymatic or microbial detoxification, referred to as “biotransformation” or “biodetoxification,” utilizes microorganisms or purified enzymes thereof to catabolize the entire mycotoxin or transform or cleave it to less or non-toxic compounds. However, the awareness on the prevalence of mycotoxins, available modern techniques to analyze them, the effects of mycotoxicoses, and the recent developments in the ways to safely eliminate the mycotoxins from the feed are very minimal among the producers. This symposium review paper comprehensively discusses the above mentioned aspects.