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32 results on '"Biochemical tests"'

1. Biochemical and molecular characterization of Campylobacter fetus isolates from bulls subjected to bovine genital campylobacteriosis diagnosis in Spain

Author

Nerea Pena-Fernández, Nekane Kortabarria, Ana Hurtado, Medelin Ocejo, Marcelo Fort, Iratxe Pérez-Cobo, Esther Collantes-Fernández, and Gorka Aduriz

Subjects
Campylobacter fetus venerealis, Biochemical tests, PCR, Whole Genome Sequencing (WGS), Bovine Genital Campylobacteriosis (BGC), Veterinary medicine, SF600-1100
Abstract

Abstract Background Bovine genital campylobacteriosis (BGC) is caused by Campylobacter fetus subsp. venerealis (Cfv) including its biovar intermedius (Cfvi). This sexually transmitted disease induces early reproductive failure causing considerable economic losses in the cattle industry. Using a collection of well-characterized isolates (n = 13), C. fetus field isolates (n = 64) and saprophytic isolates resembling Campylobacter (n = 75) obtained from smegma samples of breeding bulls, this study evaluated the concordance of the most used phenotypic (H2S production in cysteine medium and 1% glycine tolerance) and molecular (PCR) methods for the diagnosis of BGC and assessed possible cross-reactions in the molecular diagnostic methods. Results Characterization at the subspecies level (fetus vs. venerealis) of C. fetus isolated from bull preputial samples using phenotypic and molecular (PCR targeting nahE and ISCfe1) methods showed moderate concordance (κ = 0.462; CI: 0.256–0.669). No cross-reactions were observed with other saprophytic microaerophilic species or with other Campylobacter species that can be present in preputial samples. Whole genome sequencing (WGS) of discrepant isolates showed 100% agreement with PCR identification. For the differentiation of Cfv biovars, comparison of the H2S test (at 72 h and 5 days of incubation) and a PCR targeting the L-cysteine transporter genes showed higher concordance when H2S production was assessed after 5 days (72 h; κ = 0.553, 0.329–0.778 CI vs. 5 days; κ = 0.881, 0.631–1 CI), evidencing the efficacy of a longer incubation time. Conclusions This study confirmed the limitations of biochemical tests to correctly identify C. fetus subspecies and biovars. However, in the case of biovars, when extended incubation times for the H2S test (5 days) were used, phenotypic identification results were significantly improved, although PCR-based methods produced more accurate results. Perfect agreement of WGS with the PCR results and absence of cross-reactions with non-C. fetus saprophytic bacteria from the smegma demonstrated the usefulness of these methods. Nevertheless, the identification of new C. fetus subspecies-specific genes would help to improve BGC diagnosis.

Published
2024
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3. Isolation and Molecular Identification of Salmonella pullorum from Broiler Chicken in Iraqi Fields.

Author

Hawal, R. Ghazy and Bakr, M. Talib

Subjects
BROILER chickens, SALMONELLA, SALMONELLA enterica, SALMONELLA detection, SEROTYPING
Abstract

Pullorum disease (PD) is one of the most common diseases in the world, with devastating consequences. In the chicken sector, there have been financial losses. It is brought on by Salmonella enteric subspecies serovar Gallinarum biovar pullorum; definitive detection requires culture followed by biochemistry analysis and serotyping. This study aimed to verify the presence of bacteria by culture, biochemical characterization, PCR assay, and sequencing. One hundred samples were collected from 12 broiler chicken flocks of different ages for 8districts of Baghdad province, including cloacal swabs (65), visceral organs (15), and dropping (20). Salmonella colonies were identified by selective culture broth and agar with biochemical description for 75% of the total samples, with a higher incidence in visceral organs than dropping and cloacal swabs. ،The Sequencing and phylogenetic tree analysis of 16S rRNA gene for representative Salmonella isolates. The presence of Salmonella pullorum isolates in global genetic strains; was revealed a matching NCBI isolates similarity of 99.02% with (MF445124.1) and 98% with (MH352164.1), respectively. In the current state of molecular and genetic research, phlyogentic research announced the real presence of Salmonella pullorum in Baghdadprovince's broiler chicken, also showing the phylogentic characteristics and links to some global isolates. The detection of Salmonella pullorum in broiler flocks of the current study extent of health risks to other uninfected birds present in the free range. [ABSTRACT FROM AUTHOR]

Published
2023
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4. Molecular Confirmation and Immunological Cross Reactivity among Mycoplasma gallisepticum Isolates Recovered from Broiler Chicken in Khyber Pakhtunkhwa, Pakistan.

Author

Ur Rehman, Atta, Shah, Abdul Haleem, Ur Rahman, Sajjad, Sajid, Sanaullah, Khan, Imdad Ullah, Ullah, Qudrat, Farid, Arshad, and Khan, Muhammad Hashim

Subjects
*MYCOPLASMA gallisepticum, *BROILER chickens, *POULTRY industry, *IMMUNOGLOBULINS, *ENVIRONMENTAL sampling, *EGGS as food
Abstract

Mycoplasma gallisepticum (MG) is responsible for serious economic losses in poultry industry in Pakistan. The present study was designed to evaluate the molecular and immunological cross reactivity among MG isolates recovered from broiler birds. For this purpose a total of four hundred and thirty-four (n=434) specimen samples were taken from suspected broilers as well as environmental samples of forty commercial poultry (broiler) farms from DIK, Tank, Peshawar, Abbottabad and Mansehra districts of Khyber Pakhtunkhwa. These samples were inoculated and serially passage into Modified Frey's broth. One hundred and nine (25.1%) samples showed turbidity and were further processed through biochemical tests (Catalase production, Tetrazolium reduction, Phosphatase production, Film and spot Assay and Glucose fermentation). Typical fried egg colonies on Frey's agar were purified. Biochemical tests indicated Mycoplasma species as 2.5% of total specimen samples. The PCR results confirmed (185 bp) three MG isolates (MG-I, MG-II and MG-III) with 0.7% prevalence in total specimen samples. All three antigens (Formalin inactivated and Montanide adjuvanted) were subcutaneously inoculated @ 0.5 ml into rabbits and booster on 7 days post first inoculation. Sera samples collected after 14 days post booster shot, were processed for antibody titration using Haemagglutination inhibition (HI) test with homologous and heterologous antigens. The immunological cross reactivity indicated all three MG isolates as distinctly separate from each other. The immunological cross reactivity was existing with 66 to 87.18% among all three isolates. The results showed high homologous GMT titers as compared to heterologous for each MG isolate. [ABSTRACT FROM AUTHOR]

Published
2022
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5. Detection of Salmonella spp. by Traditional and PCR Assays in Raw Milk, Maysan, Iraq.

Author

Dawood Saleem, H., Fawwaz Alfarras, A., Hameed, N. M., Hasan Al-Zubaidi, S., Shnain Ali, M., Hamood, S. A., Hameed, S., Hamad, D. A., Ali Hussein, H., and Mohsin Al-Dhalemi, D.

Subjects
RAW milk, SALMONELLA detection, SALMONELLA, GRAM-negative bacteria, POLYMERASE chain reaction
Abstract

Salmonella spp are characterized as rod-shaped, motile, gram-negative bacteria which has the ability to infect animals and human. Salmonella spp occasionally causes sickness while in most cases not lead to severe symptoms. Analyzing milk for Salmonella spp. is not routine but traditional culture methods are used to evaluate the health condition of the dairy products. However, the antibody-based and nucleic-acid-based methods are practical for identifying Salmonella spp. Therefore, this research was designed to evaluate the use of traditional culture methods and PCR in detection of the presence of Salmonella spp. in raw milk samples in, Maysan Iraq. A total number of 130 raw milk samples collected from Maysan Iraq. All the samples were analyzed for the presence of Salmonella spp. using traditional culture method and polymerase chain reaction (PCR). The culture method used in this experiment were done by using pre-enrichment, enrichment, selective plating and biochemical tests. The results of this traditional technique were compared with the results obtained from PCR method. The PCR was performed using a 284bp sequence of the invA gene. The results showed that 8 (7.07%) of samples were identified as salmonella positive using traditional culture technique but 14 (12.3%) samples were detected as salmonella positive by PCR method. The results of the current research revealed that the traditional culture based methods are generally time costuming and labor intensive but the development of new rapid methods including DNA based methods such as PCR are more sensitive and have dramatically decreased the time necessary for the detection of bacteria. [ABSTRACT FROM AUTHOR]

Published
2022
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7. تشخیص بیماری لاکتوکوکوزیس در یکی از مزارع پرورش ماهی قزل‌آلای رنگین کمان (Oncorhynchus mykiss) استان خوزستان.

Author

فریدون حسنی, رحيم پيغان, طاهره عبياوی, داریوش غریبی, and تکاور محمدیان

Abstract

Lactococcus garviea is a gram positive, facultative anaerobic, oxidase and catalase negative that belong to the lactic acid group bacteria. This bacterium is the cause of Lactococcosis, an acute disease with hemorrhagic septicemia in rainbow trout and causes a lot of economic damage to the aquaculture industry. In this study, 10 diseased rainbow trout were bacteriologically sampled from head kidney and brain in one of fish farms of Khuzestan province, Located in Masjed Soleyman. The obtained isolates were further evaluated by biochemical tests and molecular method (with 16S rRNA specific primers). Samples were also taken from different organs and fixed in formalin 10% for pathological examination. To investigate the lesions, sections were prepared from 5 μm and stained with Hematoxylin-Eosin. The results of bacterial culture, biochemical tests and PCR confirmed the presence of Lactococcosis in the studied farms. Macroscopic examination revealed lesions in the liver, kidneys and intestine of affected fish. The liver was pale and the kidneys were relatively swollen. No other complications or lesions, including abdominal discharge, were observed in affected fish. In histopathologic examination of liver, degeneration, and focal necrosis were seen. In kidneys, tubules degeneration and melanomacrophage cells accumulation were seen. In intestines, moderate inflammation and inflammatory cells aggregation was seen. According to the confirmation of the presence of lactococcosis, it is necessary to take measures to prevent lactococcosis (especially vaccination). [ABSTRACT FROM AUTHOR]

Published
2022
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8. Evaluation of API 20E system in fluorescent Pseudomonas identification from button mushroom Agaricus bisporus cultivation casing soil

Author

Anwar O. MOHAMMAD, Abdulkareem E.S. ALKURTANY, and Abdullah A. HASSAN

Subjects
bacteria, biochemical tests, classification, fluorescent Pseudomonas, PCR, Agriculture (General), S1-972, Science (General), Q1-390
Abstract

Bacterial activity, mainly Pseudomonas spp. plays a vital role in the fruiting process of white button mushroom, hence a rapid procedure to identify these bacteria is crucial. In the current study, the validity of commercial identification system, Analytical profile index API 20E to identify Pseudomonas isolates from mushroom casing soil were assessed. Using API strips fifty bacterial isolates from a selective medium (King B medium) were examined, all isolates were belonged to the genus Pseudomonas according to API 20E identification systems. However, only 74% of Pseudomonas bacteria were identified to species level. The molecular identification using 16S rRNA gene was used as a reference tool to identify bacteria at the species level. The results show that the accuracy of the system to classify florescent Pseudomonas to species level was 60%. This was species dependant, and the system accuracy were 100%, 87.5%, 81.3% and 63% in identifying P. aeruginosa, P. putida, P. fluorescens and P. tolaasii respectively. Our finding indicates that although the classification of the Pseudomonas genus with API 20E system is useful, but it is not enough to distinguish these bacteria to species level, genomic studies are necessary to confirm the exact taxonomic position of Pseudomonas spp.

Published
2020
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9. Evaluation of API 20E system in fluorescent Pseudomonas identification from button mushroom Agaricus bisporus cultivation casing soil.

Author

MOHAMMAD, Anwar O., ALKURTANY, Abdulkareem E. S., and HASSAN, Abdullah A.

Subjects
*TILLAGE, *CULTIVATED mushroom, *PSEUDOMONAS, *SYSTEM identification, *MUSHROOMS
Abstract

Bacterial activity, mainly Pseudomonas spp. plays a vital role in the fruiting process of white button mushroom, hence a rapid procedure to identify these bacteria is crucial. In the current study, the validity of commercial identification system, Analytical profile index API 20E to identify Pseudomonas isolates from mushroom casing soil were assessed. Using API strips fifty bacterial isolates from a selective medium (King B medium) were examined, all isolates were belonged to the genus Pseudomonas according to API 20E identification systems. However, only 74% of Pseudomonas bacteria were identified to species level. The molecular identification using 16S rRNA gene was used as a reference tool to identify bacteria at the species level. The results show that the accuracy of the system to classify florescent Pseudomonas to species level was 60%. This was species dependant, and the system accuracy were 100%, 87.5%, 81.3% and 63% in identifying P. aeruginosa, P. putida, P. fluorescens and P. tolaasii respectively. Our finding indicates that although the classification of the Pseudomonas genus with API 20E system is useful, but it is not enough to distinguish these bacteria to species level, genomic studies are necessary to confirm the exact taxonomic position of Pseudomonas spp. [ABSTRACT FROM AUTHOR]

Published
2020
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10. MORPHOLOGICAL, BIOCHEMICAL AND GENETIC CHARACTERIZATION OF CITRUS CANKER PATHOGEN (XANTHOMONASAXONOPODIS) FROM CITRUS CULTIVARS OF PUNJAB, PAKISTAN.

Author

Shehzadi, I. and Naz, S.

Subjects
*CITRUS canker, *GRAPEFRUIT, *CULTIVARS, *GRAM'S stain, *PATHOGENIC microorganisms, *BIOMARKERS, *CITRUS
Abstract

The objective of the present study was to identify citrus canker pathogen (Xanthomonas axonopodis) and disease severity among different citrus cultivars collected from citrus growing area (CRI) of Punjab, Pakistan. Morphological, biochemical and molecular markers characterized the pathogen. Total 95 samples were collected from grapefruit (C. paradis), kumquats (C. japonica) and lemon and limes (C. limon) and subjected to biochemical analysis by API strip. Three different primer sets targeting 16SrDNA and internally transcribed spacer region (ITS) were used to check genetic variability by PCR. The infectious pathogen colonies were found to be small, yellow, mucoid and gram negative by gram staining. The oxidase, urease tests were negative and casesin hydrolysis, citrate utilization, methyl red test, catalase tests were positive for the pathogen via API strips. All the collected samples were positive for the disease by PCR and the disease severity was also measured by percentage mean. The highest disease severity (82%) was observed in grapefruits followed by lemon and limes (70%) while the lowest (30%) was in kumquats. The present results revealed that citrus cultivars have variability towards the susceptibility of citrus canker and phylogenetic analysis revealed that A type citrus canker is prevalent in selected region of Pakistan. [ABSTRACT FROM AUTHOR]

Published
2019

12. Evaluation of the presence of Vibrio parahaemolyticus in white shrimp (Litopenaeus vannamei) estuarine-wild from southern Sinaloa and northern Nayarit by microbiological analysis and PCR

Author

Méndez-Gómez, E., Rivas-Montaño, A.M., and Cortés-Ruiz, J.A.

Subjects
Shrimp, V. parahaemolyticus, biochemical tests, PCR, gastrointestinal health risk., Agriculture, Environmental sciences, GE1-350
Abstract

Given the incidence of human poisonings attributed to raw shrimp consumption in southern of Sinaloa and northern of Nayarit in recent years, white shrimp (Litopenaeus vannamei) was sampled from three wildestuaries where it’s been captured and one sample was taken from a sale center in order to determine the possible presence of toxigenic Vibrio parahaemolyticus, from May to December of 2012. Samples were analyzed by Biochemical test and Polymerase Chain Reaction (PCR), they were also tested for the specific an toxicological identification, using molecular oligo nucleotides markers tlh, tdh and trh. The number of positives results were recorded to a table of most probable number (MPN).The number of samples positive for V. parahaemolyticus, weren’t toxigenic and it was demonstrated that they were below the limit established in the Mexican Official Standard NOM-242-SSA1-2009. In conclusion, the present study revealed that consumption of raw shrimp from the dates and sites sampled did not show to be a risk for human gastrointestinal diseases.

Published
2014

13. Evaluation of API 20E system in fluorescent Pseudomonas identification from button mushroom Agaricus bisporus cultivation casing soil

Author

Abdullah A. Hassan, Abdulkareem E.S. Alkurtany, and Anwar O. Mohammad

Subjects
Soil bacteria, Mushroom, biology, Pseudomonas, Medicine (miscellaneous), bacteria, biochemical tests, classification, fluorescent Pseudomonas, PCR, Forestry, Soil classification, Plant Science, Horticulture, biology.organism_classification, lcsh:S1-972, Agricultural and Biological Sciences (miscellaneous), Biochemistry, Genetics and Molecular Biology (miscellaneous), Identification (biology), Food science, lcsh:Agriculture (General), lcsh:Science (General), Agronomy and Crop Science, Casing, Agaricus bisporus, lcsh:Q1-390
Abstract

Bacterial activity, mainly Pseudomonas spp. plays a vital role in the fruiting process of white button mushroom, hence a rapid procedure to identify these bacteria is crucial. In the current study, the validity of commercial identification system, Analytical profile index API 20E to identify Pseudomonas isolates from mushroom casing soil were assessed. Using API strips fifty bacterial isolates from a selective medium (King B medium) were examined, all isolates were belonged to the genus Pseudomonas according to API 20E identification systems. However, only 74% of Pseudomonas bacteria were identified to species level. The molecular identification using 16S rRNA gene was used as a reference tool to identify bacteria at the species level. The results show that the accuracy of the system to classify florescent Pseudomonas to species level was 60%. This was species dependant, and the system accuracy were 100%, 87.5%, 81.3% and 63% in identifying P. aeruginosa, P. putida, P. fluorescens and P. tolaasii respectively. Our finding indicates that although the classification of the Pseudomonas genus with API 20E system is useful, but it is not enough to distinguish these bacteria to species level, genomic studies are necessary to confirm the exact taxonomic position of Pseudomonas spp.

Published
2020
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14. Evaluation of a simplified key for the identification of coagulasepositive Staphylococcus isolated from bovine mastitis = Avaliação de uma chave de identificação simplificada para Staphylococcus coagulase-positivos isolados de mastite bovina

Author

Ulisses de Pádua Pereira, Nivaldo da Silva, Roberta Hilsdorf Piccoli, Demétrio Junqueira Figueiredo, Luciano Vilela Paiva, and Geraldo Márcio da Costa

Subjects
mastitis pathogens, characterization, coagulase test, PCR, biochemical tests, patógenos da mastite, caracterização, teste de coagulase, testes bioquímicos, Biology (General), QH301-705.5, Microbiology, QR1-502
Abstract

Evaluation of a simplified key for the identification of coagulase-positive Staphylococcus isolated from bovine mastitis. Three hundred fourty four strains of coagulasepositive Staphylococcus (CPS), isolated from mastitis cases, underwent phenotypic and genotypictests to evaluate the efficiency of a simplified key, based on phenotypic tests for the discrimination of these microorganisms. The tests consisted of amplification of the femA gene and hemolysis in blood agar, production of acetoin and fermentation of maltose, mannitol and trehalose. Strains that showed negative results in the amplification test of the femA gene or that were not identified as Staphylococcus aureus (S. aureus) by phenotypic tests were tested with the APISTAPH kit (Biomériux-France), for precise identification of species. Phenotypic tests revealed 338 strains (98.25%) as S. aureus, three strains (0.86%) as Staphylococcus hyicus, and threemicroorganisms (0.86%) as Staphylococcus intermedius. PCR demonstrated that 338 (98.25%) strains belonged to the S. aureus species, confirming the results for 336 strains from 338 identified, through a simplified phenotypic key. A high rate of correlation (98.83%) was verifeid between the results of genotypic and phenotypic tests for the identification of S. aureus, demonstrating the applicability of the proposed key, for the discrimination of this microorganism in CPS isolated from bovine mastitis.Visando testar a eficiência de uma chave simplificada baseada em testes fenotípicos para a discriminação de Staphylocococcus coagulasepositivos (SCP) isolados de infecções intramamárias de bovinos, 344 amostras destes microrganismos foram submetidas a testes fenotípicos e genotípicos. Estes consistiram na amplificação do gene femA, na observação de hemólise em ágar sangue, produção de acetoína efermentação de maltose, manitol e trealose. Amostras que apresentaram resultado negativo na amplificação do gene femA ou que foram identificadas com não Staphylococcus aureus (S. aureus) por meio dos testes fenotípicos foram submetidas ao kit APISTAPH (Biomériux-França) para identificação mais precisa. Os testes fenotípicos utilizados na chave simplificada permitiram identificar 338 amostras (98,25%) como S. aureus, três amostras (0,86%) como Staphylococcus hyicus e três (0,86%) como Staphylococcus intermedius. Por meio da reação em cadeia da polimerase (PCR) 338 (98,25%) amostras foram identificadas como S. aureus, ratificando os resultados para 336 das338 amostras identificadas por meio da chave fenotípica simplificada. Observou-se elevada concordância (98,83%) entre os resultados dos testes genotípicos e fenotípicos para a identificação de S. aureus, demonstrando a aplicabilidade da chave de identificação proposta para a discriminação deste microrganismo entre SCP isolados de casos de mastite bovina.

Published
2010

15. BACTERIAL STEM ROT OF GLOBE ARTICHOKE (CYNARA CARDUNCULUS var. SCOLYMUS) CAUSED BY PECTOBACTERIUM CAROTOVORUM subsp. CAROTOVORUM IN TURKEY.

Author

Ustun, N. and Arslan, N.

Subjects
CARDOON, ARTICHOKES, ERWINIA, PLANT stems, PLANT diseases, PLANT genetics
Abstract

In the autumn of 2012 unusual symptoms on globe artichoke (Cynara cardunculus var. scolymus) cultivar A 106 were observed in commercial fields in Aydın province (Şahnalı) in the Aegean Region of Turkey. Diseased plants showed stem rot and pith necrosis symptoms at the stem base and collapsed soon after first symptoms appeared. The disease prevalence was estimated to approach 10-15% in different fields, leading to removal and destruction of many plants by the growers. Representative strains isolated from infected plants were identified on the basis of morphological, biochemical, sequencing and pathogenicity tests as Pectobacterium carotovorum. The identification was further confirmed by sequencing the pel housekeeping gene of one representative isolate (GenBank accession No. KT828557), which was found to have 100% nucleotide identity with strain HNXDT002 (GenBank accession No. JF721960.1) of P. carotovorum subsp. carotovorum, isolated previously from artichoke plants with stem rot symptoms in China. To our knowledge this is the first report on bacterial stem rot of artichoke in Turkey. [ABSTRACT FROM AUTHOR]

Published
2016

16. Species level identification of thermotolerant campylobacters

Author

I. Kolackova and R. Karpiskova

Subjects
c. jejuni, c. coli, food products, biochemical tests, food borne disease, pcr, Veterinary medicine, SF600-1100
Abstract

The aim of this study was to compare the phenotypic and genotypic based methods for species identification of thermotolerant campylobacters of human and food origin from the Czech Republic. Phenotypic methods are time-consuming and sometimes lead to intermediate results, therefore replacement by more specific and rapid methods are needed. Out of a total of 911 campylobacter strains tested, 800 human isolates were received from the clinical bacteriology laboratories from 5 regions and 111 foodstuff isolates (raw chicken and pork meat from retail market) originated from the routine examination in our laboratory. Based on the PCR method 85.1% of these strains were identified as C. jejuni, 12.5% as C. coli and 2.3% as mixed cultures of C. jejuni and C. coli. When species determination of campylobacters was based on conventional methods (hippurate hydrolysis test), 28.5% of the isolates were not identified correctly. The mixed cultures of campylobacters have not been detected without further subculturing of strains, which takes several days and enormously extends the identification process. The use of the PCR method showed to be a useful tool for species identification of Campylobacter spp.

Published
2005
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17. Evaluation of a simplified key for the identification of coagulase-positive Staphylococcus isolated from bovine mastitis - doi: 10.4025/actascibiolsci.v32i4.6276

Author

Geraldo Márcio da Costa, Luciano Vilela Paiva, Roberta Hilsdorf Piccoli, Demétrio Junqueira Figueiredo, Ulisses de Pádua Pereira, and Nivaldo da Silva

Subjects
mastitis pathogens, characterization, coagulase test, PCR, biochemical tests, Biology (General), QH301-705.5, Microbiology, QR1-502
Abstract

Evaluation of a simplified key for the identification of coagulase-positive Staphylococcus isolated from bovine mastitis. Three hundred fourty four strains of coagulase-positive Staphylococcus (CPS), isolated from mastitis cases, underwent phenotypic and genotypic tests to evaluate the efficiency of a simplified key, based on phenotypic tests for the discrimination of these microorganisms. The tests consisted of amplification of the femA gene and hemolysis in blood agar, production of acetoin and fermentation of maltose, mannitol and trehalose. Strains that showed negative results in the amplification test of the femA gene or that were not identified as Staphylococcus aureus (S. aureus) by phenotypic tests were tested with the APISTAPH kit (Biomériux-France), for precise identification of species. Phenotypic tests revealed 338 strains (98.25%) as S. aureus, three strains (0.86%) as Staphylococcus hyicus, and three microorganisms (0.86%) as Staphylococcus intermedius. PCR demonstrated that 338 (98.25%) strains belonged to the S. aureus species, confirming the results for 336 strains from 338 identified, through a simplified phenotypic key. A high rate of correlation (98.83%) was verifeid between the results of genotypic and phenotypic tests for the identification of S. aureus, demonstrating the applicability of the proposed key, for the discrimination of this microorganism in CPS isolated from bovine mastitis.

Published
2010
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18. Isolation and identification of bovine Brucella isolates from Pakistan by biochemical tests and PCR.

Author

Ali, Shahzad, Ali, Qurban, Melzer, Falk, Khan, Iahtasham, Akhter, Shamim, Neubauer, Heinrich, and Jamal, Syed

Abstract

Brucellosis is endemic in bovines in Pakistan. The Brucella species and biovars involved, however, are unknown. The objectives of the present study were to isolate and characterize brucellae from seropositive milk samples, aborted fetuses, and vaginal swabs of cattle and buffaloes which had recently aborted. The seropositive milk samples, aborted fetuses, and vaginal swabs of cattle and buffaloes were collected from the Potohar Plateau, Pakistan. Isolation of brucellae was done on modified Farrell's serum dextrose agar. Isolates were characterized by conventional biotyping methods, while molecular typing was done by genus (B4/B5) and species-specific ( Brucella abortus, Brucella melitensis, Brucella ovis, and Brucella suis) polymerase chain reaction (PCR). A total of 30 isolates were recovered from milk ( n = 5), aborted fetuses ( n = 13), and vaginal swabs ( n = 12). Most isolates were from cattle (56.7 %). All of them were identified as B. abortus biovar 1 based on conventional biotyping methods and genus and species-specific PCR. This preliminary study provides the first report on the prevalence of B. abortus biovar 1 in cattle and buffaloes in Pakistan. [ABSTRACT FROM AUTHOR]

Published
2014
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19. Identification of Bifidobacterium strains from faeces of lambs

Author

Bunešová, Věra, Vlková, Eva, Killer, Jiří, Rada, Vojtěch, and Ročková, Šárka

Subjects
*BIFIDOBACTERIUM, *FECES, *YEAST extract, *MUPIROCIN, *ACETIC acid, *RAPD technique, *PROBIOTICS
Abstract

Abstract: Bifidobacteria were enumerated and isolated from the faeces of lambs aged between 25 and 40days using modified trypticase–phytone–yeast extract (TPY) agar supplemented with mupirocin and acetic acid. Nineteen strains were characterised and divided into 6 groups according their fermentation characteristics obtained by API 50 CHL and RAPID ID 32 tests and by randomly amplified polymorphic DNA (RAPD) profiles. One representative strain of each group was identified by sequencing by 16S rRNA and hsp60 genes sequencing as B. animalis ssp. animalis, B. choerinum, B. pseudolongum ssp. globosum, B. pseudocatenulatum. Examination of the DNA fingerprinting showed a genetic diversity between strains of B. animalis ssp. animalis and B. choerinum. Our result shows that some bifidobacterial species should not be host-specific or have a broad host spectrum. As bifidobacteria were determined as numerous bacterial group in the intestine of lambs on a milk diet (8.32±0.71logCFU/g, mean count±S.D., n =30), there is a reasonable presumption that bifidobacteria could be an effective probiotics for lambs. Identification techniques used in this study were found to be suitable for the classification of lamb origin bifidobacteria. [Copyright &y& Elsevier]

Published
2012
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20. DETECTION AND IDENTIFICATION METHODS AND NEW TESTS AS DEVELOPED AND USED IN THE FRAMEWORK OF COST 873 FOR BACTERIA PATHOGENIC TO STONE FRUITS AND NUTS.

Author

Palacio-Bielsa, A., Pothier, J. F., Roselló, M., Duffy, B., and López, M. M.

Subjects
PATHOGENIC microorganisms, BACTERIA, QUANTITATIVE research, PLANT protection, RESERVOIRS
Abstract

Xanthomonas arboricola pv. pruni (Xap), the causal agent of bacterial spot disease of stone fruits and almond, is regulated as a quarantine pathogen in the European Union and the European and Mediterranean Plant Protection Organization (EPPO). Xap can have an epiphytic phase and/or be latent and, consequently, it can be transmitted by different types of plant material. Effective quarantine measures require specific, sensitive and rapid methods to detect Xap in propagative material or new reservoirs. Laborious and time-consuming methods for the diagnosis of Xap are recommended in the current EPPO standard protocol. However, new several pathogen-specific PCR and quantitative realtime PCR assays have been developed that enable direct detection of Xap in symptomatic and symptomless plant samples. A concise resource of current methods for Xap detection and identification, based on assessment and development activities within the framework of COST 873, is presented. [ABSTRACT FROM AUTHOR]

Published
2012

21. CURRENT STATUS OF BACTERIAL SPOT OF STONE FRUITS AND ALMOND CAUSED BY XANTHOMONAS ARBORICOLA pv. PRUNI IN SPAIN.

Author

Roselló, M., Santiago, R., Palacio-Bielsa, A., García-Figueres, F., Montón, C., Cambra, M. A., and López, M. M.

Subjects
BACTERIAL diseases, PATHOGENIC microorganisms, FATTY acid methyl esters, ALLERGIES
Abstract

In 2002, typical symptoms of bacterial spot disease of stone fruits caused by Xanthomomas arboricola pv. pruni (Xap) were observed for the first time on Japanese plum in Badajoz (south-western Spain). During the following years, the pathogen was found in seven other eastern and northern Spanish provinces (Valencia, Alicante, Zaragoza, Huesca, Navarra, Lérida and Mallorca) affecting different cultivars of Japanese plum, nectarine, peach and almond. There are few previous reports of Xap on almond, the Spanish outbreaks constituting its first detection on this host in the European Union (EU). Identification of the pathogen was performed using biochemical tests, fatty acid methyl esters (FAME) profiles, conventional and real-time PCR, and hypersensitivity reaction on tobacco leaves. Pathogenicity was demonstrated by inoculation of young potted plants of peach, plum or almond and successful re-isolations from plants with symptoms. In areas where infected plants were found, eradication programs were set up since Xap has a quarantine status according to phytosanitary EU legislation. [ABSTRACT FROM AUTHOR]

Published
2012

22. Evaluation of a simplified key for the identification of coagulasepositive Staphylococcus isolated from bovine mastitis.

Author

da Costa, Geraldo Márcio, Paiva, Luciano Vilela, Piccoli, Roberta Hilsdorf, Figueiredo, Demétrio Junqueira, de Pádua Pereira, Ulisses, and da Silva, Nivaldo

Abstract

Copyright of Acta Scientiarum: Biological Sciences is the property of Universidade Estadual de Maringa and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Published
2010
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23. Erwinia amylovora strains from outbreaks of fire blight in Spain: phenotypic characteristics.

Author

Donat, Victoria, Biosca, Elena G., Rico, Arantza, Peñalver, Javier, Borruel, Marisa, Berra, Dionisio, Basterretxea, Txaran, Murillo, Jesús, and López, Maria M.

Subjects
*ERWINIA amylovora, *FIRE-blight, *BACTERIAL diseases of plants, *ANTIBACTERIAL agents, *POLYMERASE chain reaction
Abstract

One hundred and thirty strains of Erwinia amylovora recovered from Spanish foci of fire blight from 1995 to 2000 were characterised and compared to reference strains from different sources and origins. Their rapid identification was performed by double antibody sandwich indirect (DASI) ELISA, using specific monoclonal antibodies against E. amylovora, and molecular confirmation by PCR using primers specific to the native plasmid pEA29. The Spanish strains of E. amylovora grew on different general and selective media producing typical colonies, except one of them that was deficient in levan production, whereas none of them grew on minimal agar medium with copper sulphate and low content of asparagine. All of them were susceptible to tetracycline, streptomycin, kasugamycin and oxolinic acid. Biochemical characterisation of selected strains by API 20E system revealed a great homogeneity, with 80% of the Spanish strains showing one of the two majority API 20E profiles described for E. amylovora, and the remaining strains showing minor differences. Pathogenicity on pear fruits and hypersensitivity reaction was confirmed, but a delayed reaction was observed for two Spanish strains. This is the first characterisation of a large collection of Spanish strains of E. amylovora. [ABSTRACT FROM AUTHOR]

Published
2005
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24. Diferentes métodos para aislamiento y detección de Salmonella spp. en canales porcinas

Author

Analía Inés Etcheverría, Nora Lía Padola, Cristina Monteavaro, Rocío Colello, Grisel Ramallo, María Julia Ruiz, and Cristina Villalobo

Subjects
Salmonella spp. - PCR – Inmunocromatografía - Pruebas bioquímicas, Ciencias Veterinarias, General Medicine, Biochemical tests, SALMONELLA SPP, INMUNOCROMATOGRAFIA, Inmunocromatografía, PCR, CIENCIAS AGRÍCOLAS, Otras Ciencias Veterinarias, Salmonella spp, PRUEBAS BIOQUÍMICAS, Immunochromatography, purl.org/becyt/ford/4.3 [https], purl.org/becyt/ford/4 [https], TP248.13-248.65, Biotechnology
Abstract

La Organización Mundial de la Salud (OMS) define salmonelosis como una de las enfermedades transmitida por alimentos (ETA) de mayor casuística, ampliamente extendida en todo el mundo. La enfermedad es producida por Salmonella spp. y causa una de las zoonosis más frecuentes y de mayor impacto económico. El hombre adquiere la infección después de la ingestión de alimen-tos contaminados, aunque también puede transmitirse de persona a persona o por vía fecal-oral. Actualmente, las técnicas micro-biológicas de aislamiento convencional para detección de Salmonella spp. son establecidas por el Código Alimentario Argentino para verificar la aptitud de un producto para consumo, pero éstas requieren de 4 a 5 días para la obtención de un resultado, tiem-po que juega en contra para el productor y la conservación de dichos alimentos. Por este motivo en este trabajo se analizan los métodos de diagnóstico tradicional según Normas ISO 6579:2002 con algunas modificaciones, los métodos de inmunoensayo comerciales y la Reacción en Cadena de la Polimerasa técnica (PCR) de detección del gen invA implicado en el proceso de inva-sión de cepas patógenas. Se analizaron 60 muestras procedentes de canales porcinas destinadas a comercialización. Se detectó un 10% de Salmonella spp. Se pudo determinar que el diagnóstico molecular por PCR posee alta sensibilidad, pero no es alenta-dor el resultado que reflejan los test comerciales inmunocromatográficos ya que queda en evidencia la necesidad de alta carga microbiana para un diagnóstico certero. The World Health Organization (WHO) defines salmonellosis as one of the most important foodborne diseases, widely spread worldwide. The disease is produced by Salmonella spp. and causes one of the most frequent zoonoses and of greater economic impact. The infection is acquired after ingestion of contaminated food, although it can also be transmitted from person to person or by fecal-oral route. Currently, conventional isolation microbiological techniques for detection of Salmonella spp. are established by the Argentine Food Code to verify the suitability of a product for consumption. But microbiological techniques require 4 to 5 days to obtain a result, time that plays against the producer and the conservation of such foods. For this reason in this work we analyze the traditional diagnostic methods according to ISO standards 6579: 2002 with some modifications, the commercial immunoassay methods and the Polymerase Chain Reaction technique (PCR) detection of the invA gene involved in the process of invasion of pathogenic strains. Sixty samples from pigs destined for commercialization were analyzed. 10% of Salmonella spp. was detected. It was possible to determine that the molecular diagnosis by PCR has high sensitivity, but it is not encouraging the result that reflect the commercial immunochromatographic tests since it is evident the need of high microbial load for a correct diagnosis. Fil: Ruiz, María Julia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tandil. Centro de Investigación Veterinaria de Tandil. Universidad Nacional del Centro de la Provincia de Buenos Aires. Centro de Investigación Veterinaria de Tandil. Provincia de Buenos Aires. Gobernación. Comision de Investigaciones Científicas. Centro de Investigación Veterinaria de Tandil; Argentina. Universidad Nacional del Centro de la Provincia de Buenos Aires. Facultad de Ciencias Veterinarias. Departamento de Sanidad Animal y Medicina Preventiva. Laboratorio de Inmunoquímica y Biotecnología; Argentina Fil: Ramallo, Grisel. Universidad Nacional del Centro de la Provincia de Buenos Aires. Facultad de Ciencias Veterinarias. Departamento de Sanidad Animal y Medicina Preventiva. Laboratorio de Inmunoquímica y Biotecnología; Argentina Fil: Colello, Rocío. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tandil. Centro de Investigación Veterinaria de Tandil. Universidad Nacional del Centro de la Provincia de Buenos Aires. Centro de Investigación Veterinaria de Tandil. Provincia de Buenos Aires. Gobernación. Comision de Investigaciones Científicas. Centro de Investigación Veterinaria de Tandil; Argentina. Universidad Nacional del Centro de la Provincia de Buenos Aires. Facultad de Ciencias Veterinarias. Departamento de Sanidad Animal y Medicina Preventiva. Laboratorio de Inmunoquímica y Biotecnología; Argentina Fil: Villalobo, Maria Cristina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tandil. Centro de Investigación Veterinaria de Tandil. Universidad Nacional del Centro de la Provincia de Buenos Aires. Centro de Investigación Veterinaria de Tandil. Provincia de Buenos Aires. Gobernación. Comision de Investigaciones Científicas. Centro de Investigación Veterinaria de Tandil; Argentina. Universidad Nacional del Centro de la Provincia de Buenos Aires. Facultad de Ciencias Veterinarias. Departamento de Sanidad Animal y Medicina Preventiva. Laboratorio de Inmunoquímica y Biotecnología; Argentina Fil: Monteavaro, Cristina Esther. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tandil. Centro de Investigación Veterinaria de Tandil. Universidad Nacional del Centro de la Provincia de Buenos Aires. Centro de Investigación Veterinaria de Tandil. Provincia de Buenos Aires. Gobernación. Comision de Investigaciones Científicas. Centro de Investigación Veterinaria de Tandil; Argentina. Universidad Nacional del Centro de la Provincia de Buenos Aires. Facultad de Ciencias Veterinarias. Departamento de Sanidad Animal y Medicina Preventiva. Laboratorio de Inmunoquímica y Biotecnología; Argentina Fil: Etcheverría, Analía Inés. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tandil. Centro de Investigación Veterinaria de Tandil. Universidad Nacional del Centro de la Provincia de Buenos Aires. Centro de Investigación Veterinaria de Tandil. Provincia de Buenos Aires. Gobernación. Comision de Investigaciones Científicas. Centro de Investigación Veterinaria de Tandil; Argentina. Universidad Nacional del Centro de la Provincia de Buenos Aires. Facultad de Ciencias Veterinarias. Departamento de Sanidad Animal y Medicina Preventiva. Laboratorio de Inmunoquímica y Biotecnología; Argentina Fil: Padola, Nora Lía. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tandil. Centro de Investigación Veterinaria de Tandil. Universidad Nacional del Centro de la Provincia de Buenos Aires. Centro de Investigación Veterinaria de Tandil. Provincia de Buenos Aires. Gobernación. Comision de Investigaciones Científicas. Centro de Investigación Veterinaria de Tandil; Argentina. Universidad Nacional del Centro de la Provincia de Buenos Aires. Facultad de Ciencias Veterinarias. Departamento de Sanidad Animal y Medicina Preventiva. Laboratorio de Inmunoquímica y Biotecnología; Argentina

Published
2018

25. Evaluation of a simplified key for the identification of coagulase-positive Staphylococcus isolated from bovine mastitis - doi: 10.4025/actascibiolsci.v32i4.6276 Evaluation of a simplified key for the identification of coagulase-positive Staphylococcus isolated from bovine mastitis - doi: 10.4025/actascibiolsci.v32i4.6276

Author

Ulisses de Pádua Pereira, Demétrio Junqueira Figueiredo, Roberta Hilsdorf Piccoli, Luciano Vilela Paiva, Nivaldo da Silva, and Geraldo Márcio da Costa

Subjects
PCR, lcsh:Biology (General), lcsh:QR1-502, biochemical tests, characterization, coagulase test, lcsh:QH301-705.5, lcsh:Microbiology, mastitis pathogens
Abstract

Evaluation of a simplified key for the identification of coagulase-positive Staphylococcus isolated from bovine mastitis. Three hundred fourty four strains of coagulase-positive Staphylococcus (CPS), isolated from mastitis cases, underwent phenotypic and genotypic tests to evaluate the efficiency of a simplified key, based on phenotypic tests for the discrimination of these microorganisms. The tests consisted of amplification of the femA gene and hemolysis in blood agar, production of acetoin and fermentation of maltose, mannitol and trehalose. Strains that showed negative results in the amplification test of the femA gene or that were not identified as Staphylococcus aureus (S. aureus) by phenotypic tests were tested with the APISTAPH kit (Biomériux-France), for precise identification of species. Phenotypic tests revealed 338 strains (98.25%) as S. aureus, three strains (0.86%) as Staphylococcus hyicus, and three microorganisms (0.86%) as Staphylococcus intermedius. PCR demonstrated that 338 (98.25%) strains belonged to the S. aureus species, confirming the results for 336 strains from 338 identified, through a simplified phenotypic key. A high rate of correlation (98.83%) was verifeid between the results of genotypic and phenotypic tests for the identification of S. aureus, demonstrating the applicability of the proposed key, for the discrimination of this microorganism in CPS isolated from bovine mastitis.Evaluation of a simplified key for the identification of coagulase-positive Staphylococcus isolated from bovine mastitis. Three hundred fourty four strains of coagulase-positive Staphylococcus (CPS), isolated from mastitis cases, underwent phenotypic and genotypic tests to evaluate the efficiency of a simplified key, based on phenotypic tests for the discrimination of these microorganisms. The tests consisted of amplification of the femA gene and hemolysis in blood agar, production of acetoin and fermentation of maltose, mannitol and trehalose. Strains that showed negative results in the amplification test of the femA gene or that were not identified as Staphylococcus aureus (S. aureus) by phenotypic tests were tested with the APISTAPH kit (Biomériux-France), for precise identification of species. Phenotypic tests revealed 338 strains (98.25%) as S. aureus, three strains (0.86%) as Staphylococcus hyicus, and three microorganisms (0.86%) as Staphylococcus intermedius. PCR demonstrated that 338 (98.25%) strains belonged to the S. aureus species, confirming the results for 336 strains from 338 identified, through a simplified phenotypic key. A high rate of correlation (98.83%) was verifeid between the results of genotypic and phenotypic tests for the identification of S. aureus, demonstrating the applicability of the proposed key, for the discrimination of this microorganism in CPS isolated from bovine mastitis.

Published
2010

26. Assessment of MALDI TOF MS as alternative tool for Streptococcus suis identification

Author

Marta Pérez-Sancho, Teresa García-Seco, Marcelo Gottschalk, Ana I. Vela, Lucas Domínguez, and José F. Fernández-Garayzábal

Subjects
Serotype, Veterinary medicine, Desorption ionization, Streptococcus suis, 03 medical and health sciences, biochemical markers, Medicine, Species identification, MALDI-TOF MS, Biochemical markers, 030304 developmental biology, Original Research, 0303 health sciences, biology, IDENTIFICATION, 030306 microbiology, business.industry, lcsh:Public aspects of medicine, Public Health, Environmental and Occupational Health, biochemical tests, lcsh:RA1-1270, Gold standard (test), biology.organism_classification, Matrix-assisted laser desorption/ionization, PCR, Public Health, Veterinaria, business
Abstract

The accuracy of matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) for identifying Streptococcus suis isolates obtained from pigs, wild animals, and humans was evaluated using a PCR-based identification assay as the gold standard. In addition, MALDI-TOF MS was compared with the commercial multi-tests Rapid ID 32 STREP system. From the 129 S. suis isolates included in the study and identified by the molecular method, only 31 isolates (24.03%) had score values ≥2.300 and 79 isolates (61.24%) gave score values between 2.299 and 2.000. After updating the currently available S. suis MALDI Biotyper database with the spectra of three additional clinical isolates of serotypes 2, 7, and 9, most isolates had statistically significant higher score values (mean score: 2.65) than those obtained using the original database (mean score: 2.182). Considering the results of the present study, we suggest using a less restrictive threshold score of ≥2.000 for reliable species identification of S. suis. According to this cut-off value, a total of 125 S. suis isolates (96.9%) were correctly identified using the updated database. These data indicate an excellent performance of MALDI-TOF MS for the identification of S. suis.

Published
2015
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27. Percloreto de ferro como sinalizador de injúrias em tubérculos de batata e caracterização de isolados de Pectobacterium spp

Author

Adelaide Silva, Tebaldi, Nilvanira Donizete, Luz, José Magno Queiroz, Soratto, Rogério Peres, Queiroz, Angélica Araújo, and Vieira, Carlos Ueira

Subjects
PCR, Testes bioquímicos, Batata, Transport, CIENCIAS AGRARIAS::AGRONOMIA [CNPQ], Rating scale, Transporte, Biochemical tests, Solanum tuberosum, Batata - Armazenamento, Percloreto de ferro
Abstract

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior The use of iron perchlorate as a marker of injury in potato tubers may help preventloss of lots during storage and transport. The objectives of this study were to determine the concentration of iron perchlorate solution and the immersion time for the marking of pectolytic bacteria in potato tubers; the evaluation of potato batches for the presence of these bacteria; the biochemical, physiological and molecular characterization of Pectobacterium spp. isolates; and the indication of pectobacteria by iron perchlorate in inoculated and commercial batches of potato tubers. The experiments were done at the Universidade Federal de Uberlândia, from 2009 to 2011. Marking of pectolytic bacteria in potato tubers was done using iron perchlorate concentrations of 0, 8, 21 and 44%, in which a sample of 10 tubers were immersed for 10, 20 or 30 minutes, and evaluated after 10, 20 and 30 minutes at room temperature, evaluating the appearance dark spots on the tubers. The isolates were characterized culturally, biochemically and physiologically by the tests of: Gram, glucose fermentation, catalase, oxidase, growth at 37ºC, reduction of substances from sucrose, phosphatase activity, indol production and acid production from sorbitol, melibiose, citrate or lactose, hypersensibility reaction in tobacco and pathogenicity in potato baits. Molecular characterization was done using the primers: EXPCCR/EXPCCF; ECA1f/ECA2r, 1491f, L1RA/L1RG Br1f. The indication of pectobacteria by the product studied in inoculated seed potato tubers was done with the cultivars Ágata and Atlantic. The tubers were perforated, or not, with a needle and immersed in distilled water or in bacterial suspension (isolate UFU A14 of Pectobacterium spp.) for 5 minutes and incubated at different temperatures (22, 28 or 35°C) and times (0, 24, 48, 72 or 96 hours), and subsequently immersed in the de perchlorate solution for the evaluation of lesion diameter. The experimental design was randomized blocks with four repetitions, as split plots in space 5 x 4. The indication of pectobacteria by the product in commercial potato batches was done with cultivars Ágata and Asterix. The samples with six tubers were evaluated 12 and 24 hours after harvest and incubated in five temperatures (8, 18, 28, 38 or 48°C), and immersed in 8% iron perchlorate solution for 10 minutes, removed and left at room temperature for 20 minutes before evaluation with a rating scale, varying from 0 to 4. Immersion for 10 minutes in iron perchlorate at 21% and evaluation after 20 minutes at room temperature was adequate to indicate the presence of pectobacteria. The isolates could not be characterized biochemistry and molecular. The inoculation test was able to confirm the presence of pectobacteria in the tubers by perchlorate. Storage and transport of commercial potatoes should be done at lower temperatures; the cultivar Ágata was more susceptible than Asterix to the pectobacteria. O uso de percloreto de ferro como sinalizador de injúrias em tubérculos de batata poderá ajudar evitando perdas de lotes durante o armazenamento e transporte. Os objetivos do trabalho foram determinar a concentração da solução de percloreto de ferro e tempo de imersão para a sinalização de bactérias pectolíticas em tubérculos de batata; a qualificação de lotes de batata quanto à presença destas bactérias; a caracterização bioquímica, fisiológica e molecular de isolados de Pectobacterium spp. e indicação de pectobactérias por percloreto de ferro em tubérculos de batata inoculados e lotes comerciais. Os experimentos foram conduzidos na Universidade Federal de Uberlândia, entre os anos de 2009 e 2011. Para a sinalização de bactérias pectolíticas em tubérculos de batata, foram utilizadas as concentrações 0,0; 8,4; 21,0 e 44,0% de percloreto de ferro, em que amostras de 10 tubérculos ficaram imersas por 10, 20 e 30 minutos. Em seguida, avaliou-se, aos 10, 20 e 30 minutos e em temperatura ambiente, o surgimento de manchas escuras nos tubérculos. Os isolados foram caracterizados cultural, bioquímica e fisiologicamente pelos testes: Gram, fermentação de glicose, catalase, oxidase, crescimento à 37ºC, redução de substâncias a partir de sacarose, atividade da fosfatase, produção de indol e produção de ácido a partir de sorbitol, melibiose, citrato e lactose, reação de hipersensibilidade em fumo e patogenicidade em isca de batata. Para a caracterização molecular, foram utilizados os primers: EXPCCR/EXPCCF; ECA1f/ECA2r, 1491f, L1RA/L1RG Br1f. Para a indicação de pectobactérias pelo produto estudado em tubérculos de batata-semente inoculados, utilizou-se as cvs. Ágata e Atlantic. Os tubérculos foram perfurados e não perfurados com estilete e imersos em água destilada ou na suspensão bacteriana (isolado UFU A14 de Pectobacterium spp.) por 5 minutos e incubados em diferentes temperaturas (22°C, 28°C e 35°C) e tempos (0, 24, 48, 72 e 96 horas); em seguida, imersos em solução de percloreto de ferro com avaliação do diâmetro da lesão formada. O delineamento foi em blocos casualizados com quatro repetições, em esquema de parcelas subdivididas no espaço 5 x 4. Para a indicação de pectobactérias pelo produto em lotes comerciais de batata, foram utilizadas as cultivares Ágata e Asterix. As amostras com seis tubérculos foram avaliadas as 12 e 24 horas após a coleta e incubadas em cinco temperaturas (8°C, 18°C, 28°C, 38°C e 48°C), em seguida, foram imersas por 10 minutos na solução de percloreto de ferro a 8%, retiradas e deixadas em temperatura ambiente por 20 minutos para avaliação através de uma escala de notas variando de 0 a 4. Os tubérculos de batatas foram imersos por 10 minutos na solução de percloreto de ferro 21% e avaliados após 20 minutos no ambiente o que foi suficiente para indicar a presença de fitobactérias. Os isolados não puderam ser caracterizados bioquimica e molecularmente. O teste de inoculação conseguiu comprovar a presença de pectobactérias nos tubérculos pelo percloreto de ferro. Para os lotes comerciais, as menores temperaturas são mais indicadas para o armazenamento e transporte, e a cv. Ágata foi mais suscetível que Asterix. Doutor em Agronomia

Published
2012

28. Current status of bacterial spot of stone fruits and almond caused by Xanthomonas arboricola pv. pruni in Spain

Author

Roselló Pérez, Montserrat, Santiago, R., Palacio Bielsa, Ana, García Figueres, F., Montón, C., Cambra Alvarez, Miguel Angel, and López, M.M.

Subjects
PCR, Inoculation, Sanidad vegetal, Diagnosis, España, food and beverages, Árboles frutales, Symptomatology, Biochemical tests, Organismos patógenos, FAME, Enfermedades de las plantas
Abstract

In 2002, typical symptoms of bacterial spot disease of stone fruits caused by Xanthomomas arboricola pv. pruni (Xap) were observed for the first time on Japanese plum in Badajoz (south-western Spain). During the following years, the pathogen was found in seven other eastern and northern Spanish provinces (Valencia, Alicante, Zaragoza, Huesca, Navarra, Lérida and Mallorca) affecting different cultivars of Japanese plum, nectarine, peach and almond. There are few previous reports of Xap on almond, the Spanish outbreaks constituting its first detection on this host in the European Union (EU). Identification of the pathogen was performed using biochemical tests, fatty acid methyl esters (FAME) profiles, conventional and real-time PCR, and hypersensitivity reaction on tobacco leaves. Pathogenicity was demonstrated by inoculation of young potted plants of peach, plum or almond and successful re-isolations from plants with symptoms. In areas where infected plants were found, eradication programs were set up since Xap has a quarantine status according to phytosanitary EU legislation. Published

Published
2012

29. Detection and identification methods and new tests as developed and used in the framework of cost 873 for bacteria pathogenic to stone fruits and nuts

Author

Palacio Bielsa, Ana, Pothier, J.F., Roselló Pérez, Montserrat, Duffy, B., and López, M.M.

Subjects
PCR, Sanidad vegetal, Métodos de control, Pathogenicity tests, food and beverages, MALDI-TOF MS, Árboles frutales, Biochemical tests, Organismos patógenos, FAME, Real-time PCR, Isolation
Abstract

Xanthomonas arboricola pv. pruni (Xap), the causal agent of bacterial spot disease of stone fruits and almond, is regulated as a quarantine pathogen in the European Union and the European and Mediterranean Plant Protection Organization (EPPO). Xap can have an epiphytic phase and/or be latent and, consequently, it can be transmitted by different types of plant material. Effective quarantine measures require specific, sensitive and rapid methods to detect Xap in propagative material or new reservoirs. Laborious and time-consuming methods for the diagnosis of Xap are recommended in the current EPPO standard protocol. However, new several pathogen-specific PCR and quantitative realtime PCR assays have been developed that enable direct detection of Xap in symptomatic and symptomless plant samples. A concise resource of current methods for Xap detection and identification, based on assessment and development activities within the framework of COST 873, is presented.

Published
2012

30. MOLECULAR DIAGNOSIS OF XANTHOMONAS ALBILINEANS (ASHBY) DOWSON IN VENEZUELA, THE CAUSAL AGENT OF LEAF SCALD OF SUGARCANE ¹

Author

Alvez, Beatriz, Carballo, Jeismar, Alonso, Guillermina, and Oropeza, Maira

Subjects
Saccharum sp, PCR, genotipificación, biochemical tests, pruebas bioquímicas, genotipi fication
Abstract

La escaldadura foliar (EF) causada por Xanthomonas albilineans (Ashby) Dowson, es una de las enfermedades de mayor importancia en el cultivo de la caña de azúcar, Saccharum sp., ocasionando pérdidas estimadas entre 90-100% de la producción. El uso de la reacción en cadena de la polimerasa (por sus siglas en inglés pcR) como herramienta de diagnóstico de fitopa tógenos se incrementó a nivel mundial dada su rapidez, sensi bilidad y especificidad. Dada la importancia que tiene esta enfermedad en Venezuela, se llevó a cabo el diagnóstico de la bacteria a partir de plantas de caña de azúcar sintomáticas, empleando pruebas microbiológicas, bioquí micas y moleculares. Utilizando diversos métodos de purifi cación se obtuvieron aislados bacterianos, los cuales se some tieron a análisis fenotípicos y bioquímicos, así como a ensayos de pcR con iniciadores espe cíficos y las secuencias consenso repetitivas intragénicas de entero bacterias (siglas en inglés ERIc-pcR). con el método de macerado de hojas se obtuvieron 50 aislados, de los cuales 14 se selec cionaron por pruebas microbio lógicas y bioquí micas, de estos, sólo tres cepas fueron identi ficadas como X. albilineans. Este resultado se corroboró al obtener por pcR la banda esperada de 360 pb con los iniciadores específicos sólo en estos tres aislados. Finalmente, con la técnica de genotipificación ERIC-PCR se obtuvieron patrones de bandas para las bacterias identificadas como X. albilineans que no son similares a los descritos por otros autores en otras regiones del mundo. Este trabajo representa la primera genotipificación de aislados de X. albilineans en Venezuela Leaf scald caused by Xanthomonas albilineans, is one of the most important diseases in sugarcane, Saccharum sp., which causes serious economical losses estimated between 90-100%. The use of pcR as a diagnostic tool for plant pathogens has increased because of its speed, sensitivity and specificity. Given the importance of leaf scald in Venezuela, in this work we carried out the diagnosis of X. albilineans from symptomatic sugarcane plants, using microbiological, biochemical and molecular methods. Bacterial isolates were obtained using different methods of purification. These isolates were then subjected to phenotypic and biochemical analysis, PCR with specific primers and ERIc pcR. With the method of macerated leaves 50 isolates were obtained, out of which 14 were selected for biochemical tests. From these 14 isolates only three strains were identified as X. albilineans by microbiological and biochemical tests. These results were confirmed by PCR by obtaining the expected band of 360 bp using specific primers. Finally, the ERIc-pcR banding patterns obtained for the isolates identified as X. albilineans were found to be different to those described by other authors in other regions. This work represents the first genotyping assay of isolates of X. albilineans in Venezuela

Published
2011

31. Evaluation of a simplified key for the identification of coagulase-positive Staphylococcus isolated from bovine mastitis - doi: 10.4025/actascibiolsci.v32i4.6276

Author

Costa, Geraldo Márcio da, Paiva, Luciano Vilela, Piccoli, Roberta Hilsdorf, Figueiredo, Demétrio Junqueira, Pereira, Ulisses de Pádua, and Silva, Nivaldo da

Subjects
PCR, Doenças Infecciosas dos Animais, biochemical tests, characterization, coagulase test, mastitis pathogens
Abstract

Evaluation of a simplified key for the identification of coagulase-positive Staphylococcus isolated from bovine mastitis. Three hundred fourty four strains of coagulase-positive Staphylococcus (CPS), isolated from mastitis cases, underwent phenotypic and genotypic tests to evaluate the efficiency of a simplified key, based on phenotypic tests for the discrimination of these microorganisms. The tests consisted of amplification of the femA gene and hemolysis in blood agar, production of acetoin and fermentation of maltose, mannitol and trehalose. Strains that showed negative results in the amplification test of the femA gene or that were not identified as Staphylococcus aureus (S. aureus) by phenotypic tests were tested with the APISTAPH kit (Biomériux-France), for precise identification of species. Phenotypic tests revealed 338 strains (98.25%) as S. aureus, three strains (0.86%) as Staphylococcus hyicus, and three microorganisms (0.86%) as Staphylococcus intermedius. PCR demonstrated that 338 (98.25%) strains belonged to the S. aureus species, confirming the results for 336 strains from 338 identified, through a simplified phenotypic key. A high rate of correlation (98.83%) was verifeid between the results of genotypic and phenotypic tests for the identification of S. aureus, demonstrating the applicability of the proposed key, for the discrimination of this microorganism in CPS isolated from bovine mastitis. Evaluation of a simplified key for the identification of coagulase-positive Staphylococcus isolated from bovine mastitis. Three hundred fourty four strains of coagulase-positive Staphylococcus (CPS), isolated from mastitis cases, underwent phenotypic and genotypic tests to evaluate the efficiency of a simplified key, based on phenotypic tests for the discrimination of these microorganisms. The tests consisted of amplification of the femA gene and hemolysis in blood agar, production of acetoin and fermentation of maltose, mannitol and trehalose. Strains that showed negative results in the amplification test of the femA gene or that were not identified as Staphylococcus aureus (S. aureus) by phenotypic tests were tested with the APISTAPH kit (Biomériux-France), for precise identification of species. Phenotypic tests revealed 338 strains (98.25%) as S. aureus, three strains (0.86%) as Staphylococcus hyicus, and three microorganisms (0.86%) as Staphylococcus intermedius. PCR demonstrated that 338 (98.25%) strains belonged to the S. aureus species, confirming the results for 336 strains from 338 identified, through a simplified phenotypic key. A high rate of correlation (98.83%) was verifeid between the results of genotypic and phenotypic tests for the identification of S. aureus, demonstrating the applicability of the proposed key, for the discrimination of this microorganism in CPS isolated from bovine mastitis.

Published
2010

32. Identificación y caracterización de Aeromonas sp patógenas aisladas de truchas arcoíris (Oncorhynchus mykiss) clínicamente enfermas cultivadas en piscigranjas del Perú

Author

O. Mauro Estrella, E. Felipe Ramos, O. Jeferson Nuñure, A Adhemir Valera, S Alberto Manchego, C Nieves Sandoval, and R Antonio Herrera

Subjects
General Veterinary, animal diseases, biochemical tests, Biology, biology.organism_classification, pruebas bioquímicas, Microbiology, Perú, PCR, Aeromonas, Oncorhynchus mykiss, Peru, Rainbow trout
Abstract

The present research aimed at the biochemical characterization and molecular identification of pathogenic strains of Aeromonas sp, isolated from juvenile rainbow trout with clinical signs compatible with aeromoniasis in six fish farms in Junín (2), Pasco (2), Cajamarca (1) and Ancash (1), Peru. In total, 60 juvenile trout of 4-5 months were selected, with suggestive signs of outbreaks of aeromoniasis between 2017 and 2018. Euthanasia and necropsy were performed and the external and internal lesions present were recorded. Bacterial isolation of spleen and anterior kidney samples was carried out on TSA and GSP agar for 24-48 hours at 25 °C, bacterial colonies were characterized by biochemical tests and identified by conventional PCR using specific primers of the 16S rRNA gene (Aeromonas spp.), fstA (A. salmonicida) and gyrB (A. hydrophila), and sequencing was performed and confirmed by BLAST analysis (NCBI). The external lesions were melanosis, necrotic and haemorrhagic fins, skin ulcers and abdominal distention. Internal lesions were hepatomegaly, splenomegaly, and petechial haemorrhage in the liver, pyloric cecum, and peritoneal fat. Twelve strains were isolated in GSP (7 from Junín and 5 from Cajamarca), characterized as Aeromonas spp, being negative in the Pasco and Ancash samples. The biochemical tests determined four strains as Aeromonas salmonicida and eight as mobile Aeromonas sp, differing by motility and indole tests. Molecular identification by PCR determined four A. salmonicida strains from Junín, one A. hydrophila strain from Cajamarca and seven Aeromonas spp mobile strains (3 from Junín and 4 from Cajamarca) that were negative to the PCR of A. salmonicida and A. hydrophila. BLAST analysis (NCBI) confirmed the strains A. salmonicida and Aeromonas spp (98-100% identity). Furthermore, the Cajamarca strain (identified as A. hydrophila by biochemistry) presented 95.65% identity with A. hydrophila subspecies dhakensis. In conclusion, Aeromonas salmonicida, A. hydrophila subspecies dhakensis and other pathogenic mobile Aeromonas produce symptoms associated with aeromoniasis in juvenile rainbow trout in the Junín and Cajamarca fish farms. It is the first time that A. hydrophila subsp. dhakensis is detected in the country., La investigación tuvo como objetivo la caracterización bioquímica e identificación molecular de cepas patógenas de Aeromonas sp aisladas de truchas arcoíris juveniles con signología compatible a aeromoniasis, cultivadas en seis piscigranjas de los departamentos de Junín (2), Pasco (2), Cajamarca (1) y Ancash (1), Perú. Se seleccionaron 60 truchas juveniles de 4-5 meses, con signología sugerente de brotes de aeromoniasis entre 2017 y 2018. Se realizó la eutanasia y necropsia y se registraron las lesiones externas e internas presentes. Se efectuó el aislamiento bacteriano de muestras de bazo y riñón anterior en agar TSA y GSP por 24-48 horas a 25 °C, se caracterizaron las colonias bacterianas mediante pruebas bioquímicas y se identificaron por PCR convencional utilizando cebadores específicos del gen ARNr 16S (Aeromonas spp.), fstA (A. salmonicida) y gyrB (A. hydrophila). Además, se realizó la secuenciación y se confirmó por análisis BLAST (NCBI). Las lesiones externas registradas fueron melanosis, aletas necróticas y hemorrágicas, úlceras en la piel y distensión abdominal. Las lesiones internas fueron hepatomegalia, esplenomegalia y hemorragia petequial en hígado, ciegos pilóricos y grasa peritoneal. Se aislaron 12 cepas en GSP (7 de Junín y 5 de Cajamarca), caracterizadas como Aeromonas spp, siendo negativas en las muestras de Pasco y Ancash. Las pruebas bioquímicas determinaron cuatro cepas como Aeromonas salmonicida y ocho como Aeromonas sp móviles, diferenciándose por pruebas de motilidad e indol. La identificación molecular por PCR determinó cuatro cepas A. salmonicida procedentes de Junín, una cepa A. hydrophila de Cajamarca y siete cepas móviles Aeromonas spp (3 de Junín y 4 de Cajamarca) que fueron negativas a la PCR de A. salmonicida y A. hydrophila. El análisis BLAST (NCBI) confirmó las cepas A. salmonicida y Aeromonas spp (identidad de 98-100%); además, la cepa de Cajamarca (identificada como A. hydrophila por bioquímica) presentó identidad de 95.65% con A. hydrophila subespecie dhakensis. En conclusión, Aeromonas salmonicida, A. hydrophila subespecie dhakensis y otras Aeromonas móviles patógenas causan signología asociada a aeromoniasis en truchas arcoiris juveniles en las piscigranjas de Junín y Cajamarca. Es la primera vez que se detecta A. hydrophila subsp. dhakensis en el país.

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