scholarly journals A Comparison of Peripheral Blood Smears, Autologous Cell Cultures, and Reverse Line Blot Hybridisation in Screening for Anaplasma/Ehrlichia in Roaming Dogs and Symptomatic Dogs in Trinidad

Pathogens ◽  
2021 ◽  
Vol 10 (11) ◽  
pp. 1431
Author(s):  
Karla Georges ◽  
Chuckwudozi Ezeokoli ◽  
Godwin Isitor ◽  
Alex Mutani ◽  
Olivier Sparagano ◽  
...  

This study compared two methods to detect cases of canine ehrlichiosis in a field setting. One method was a polymerase chain reaction for the 16S rRNA gene followed by reverse line blot hybridisation with genera and species-specific probes for Anaplasma/Ehrlichia. The second method was an autologous cell culture of peripheral leucocytes isolated from heparinised blood and maintained in a homologous canine serum in Dulbecco’s Modified Eagle medium without antibiotics. The cultures were examined under light microscopy for inclusion bodies after 48 h. Leucocytes were successfully propagated for 20 of the 34 samples submitted for autologous cell culture. Inclusion bodies were observed after cell culture in leucocytes of eight dogs. Two dogs were positive to the Anaplasma/Ehrlichia genera probe and six dogs were positive to the E. canis probe after reverse line blot hybridisation. There was acceptable agreement between reverse line blot hybridisation and cell culture results. Both reverse line blot hybridisation and autologous cell cultures can be used to detect E. canis in subclinical and clinical cases of disease. A definitive diagnosis of E. canis is best achieved by a combination of clinical signs, positive autologous cell culture, and reverse line blot hybridisation results.

2001 ◽  
Vol 67 (7) ◽  
pp. 3195-3200 ◽  
Author(s):  
Fanrong Kong ◽  
Gregory James ◽  
Susanna Gordon ◽  
Anna Zelynski ◽  
Gwendolyn L. Gilbert

ABSTRACT Mycoplasma arginini, M. fermentans, M. hyorhinis, M. orale, and Acholeplasma laidlawii are the members of the class Mollicutes most commonly found in contaminated cell cultures. Previous studies have shown that the published PCR primer pairs designed to detect mollicutes in cell cultures are not entirely specific. The 16S rRNA gene, the 16S-23S rRNA intergenic spacer region, and the 5′ end of the 23S rRNA gene, as a whole, are promising targets for design of mollicute species-specific primer pairs. We analyzed the 16S rRNA genes, the 16S-23S rRNA intergenic spacer regions, and the 5′ end of the 23S rRNA genes of these mollicutes and developed PCR methods for species identification based on these regions. Using high melting temperatures, we developed a rapid-cycle PCR for detection and identification of contaminant mollicutes. Previously published, putative mollicute-specific primers amplified DNA from 73 contaminated cell lines, but the presence of mollicutes was confirmed by species-specific PCR in only 60. Sequences of the remaining 13 amplicons were identified as those of gram-positive bacterial species. Species-specific PCR primers are needed to confirm the presence of mollicutes in specimens and for identification, if required.


2018 ◽  
Vol 46 ◽  
pp. 6
Author(s):  
Julia Gabriela Wronski ◽  
Bianca Santana Cecco ◽  
Luan Cleber Henker ◽  
Marina Paula Lorenzett ◽  
Paulo Michel Roehe ◽  
...  

Background: Herpetic meningoencephalitis is an infectious contagious disease worldwide distributed, most often caused by bovine alphaherpesvirus type 5 (BoHV-5), although bovine alphaherpesvirus type 1 (BoHV-1) may occasionally be the causative agent. The disease is characterized by subacute to acute clinical onset, often affecting animals submitted to stressful situations. Clinical signs are mainly neurologic due to meningoencephalitis and cortical necrosis. The involvement of the spinal cord has also been reported, however in BoHV-1 associated disease only. The aim of this report is to describe an outbreak of bovine meningoencephalomyelitis associated to BoHV-5.Case: In August 2017, nine 1-year-old calves died in a beef cattle farm with a flock of approximately 400 bovines. The animals presented neurological clinical signs characterized by excessive salivation, nasal and ocular discharges, incoordination, apathy, head tremors, head pressing, wide-based stance, recumbency followed by convulsions and paddling. According to the owner and referring veterinarian, affected animals displayed severe clinical signs with rapid progression and often leading to death in up to seven days. Four of these calves were submitted for necropsy, and gross lesions were present in the brain, characterized by mild to moderate multifocal hemorrhagic and soft areas. On cut surface, extensive areas of dark brown discoloration and malacia were observed. Histologically, lesions were characterized by extensive areas of liquefactive necrosis in the cerebral cortex grey matter, associated with inflammatory infiltrates composed of neutrophils, lymphocytes, plasma cells and foamy macrophages, as well as multifocal to coalescing areas of hemorrhage and fibrin deposition. Intranuclear eosinophilic inclusion bodies were rarely observed in neurons and astrocytes. On leptomeninges, there was diffuse inflammatory infiltrates of lymphocytes and plasma cells. Inflammation was also seen in a milder degree in the spinal cord, characterized by infiltrate of lymphocytes at grey matter, mainly around vessels. A herpesvirus which induced typical cytopathic effect in cell cultures was recovered from tissues. The isolated virus was typed as BoHV-5 by nucleotide sequencing analysis of the gC coding region.Discussion: The diagnosis of meningoencephalomyelitis associated to BoHV-5 was based on epidemiological, clinical, macroscopical, histological, virological and genomic findings. In the outbreak here reported, the disease occurred in young animals, with low morbidity but high lethality rates. Clinical signs on this case were consistent with previous reports on the literature. Bovines affected by BoHV-5 may display no gross lesions within the CNS; however, inflammatory and degenerative changes are frequently seen, characterized by malacia, leptomeningeal vessels hyperemia, edema and hemorrhages. Histologically, non-suppurative necrotizing meningoencephalitis is seen, with perivascular inflammatory infiltrates and, occasionally, intranuclear eosinophilic inclusion bodies in astrocytes and neurons. Similar but milder lesions were seen in the spinal cords of two of the necropsied calves, a feature which has only been previously associated to BoHV-1 infections. The identification of the implicated agent was accomplished by virus isolation in cell cultures followed by genome typing. Specific treatments for this condition are not currently available, and the number of animals that recover from clinically apparent disease is extremely low. Preventive measures are based on serological testing of herds, and segregation or elimination of seropositive calves. To avoid progression of the disease in seropositive animals, control efforts must be directed to avoid stressful conditions. Vaccination with BoHV-1 and BoHV-5 vaccines is expected to confer protection to clinical disease.


1999 ◽  
Vol 37 (6) ◽  
pp. 1782-1789 ◽  
Author(s):  
J. M. Gubbels ◽  
A. P. de Vos ◽  
M. van der Weide ◽  
J. Viseras ◽  
L. M. Schouls ◽  
...  

A reverse line blot (RLB) assay was developed for the identification of cattle carrying different species ofTheileria and Babesia simultaneously. We included Theileria annulata, T. parva, T. mutans, T. taurotragi, and T. velifera in the assay, as well as parasites belonging to theT. sergenti-T. buffeli-T. orientalis group. TheBabesia species included were Babesia bovis,B. bigemina, and B. divergens. The assay employs one set of primers for specific amplification of the rRNA gene V4 hypervariable regions of all Theileria andBabesia species. PCR products obtained from blood samples were hybridized to a membrane onto which nine species-specific oligonucleotides were covalently linked. Cross-reactions were not observed between any of the tested species. No DNA sequences fromBos taurus or other hemoparasites (Trypanosomaspecies, Cowdria ruminantium, Anaplasma marginale, and Ehrlichia species) were amplified. The sensitivity of the assay was determined at 0.000001% parasitemia, enabling detection of the carrier state of most parasites. Mixed DNAs from five different parasites were correctly identified. Moreover, blood samples from cattle experimentally infected with two different parasites reacted only with the corresponding species-specific oligonucleotides. Finally, RLB was used to screen blood samples collected from carrier cattle in two regions of Spain. T. annulata, T. orientalis, and B. bigeminawere identified in these samples. In conclusion, the RLB is a versatile technique for simultaneous detection of all bovine tick-borne protozoan parasites. We recommend its use for integrated epidemiological monitoring of tick-borne disease, since RLB can also be used for screening ticks and can easily be expanded to include additional hemoparasite species.


PeerJ ◽  
2020 ◽  
Vol 8 ◽  
pp. e9080
Author(s):  
Lubov Chernogor ◽  
Elizaveta Klimenko ◽  
Igor Khanaev ◽  
Sergei Belikov

Endemic sponges (Demosponges, Lubomirskiidae) dominate the fauna of the littoral zone of Lake Baikal. These freshwater sponges live in symbiosis with diverse eukaryotes and prokaryotes, including chlorophyll-containing microalgae. Within the last 5 years, the incidence of sponge disease and mortality events in Lake Baikal has increased. The etiology and ecology of these events remain unknown, in part because of the lack of models to study sponge-microbe interactions. In this work, we tested the use of primmorph cell cultures of Lubomirskia baicalensis as a tool for investigating the microbiomes of sponges. We infected primmorphs, cultured in vitro, with samples from diseased sponges and observed, by microscopy, disease symptoms, including loss of green symbionts, associated with mass die-off events. Subsequent sequencing of 16S rRNA gene fragments revealed that the microbiome community of healthy sponge and primmorphs formed a group separate from the community of diseased sponges and infected primmorphs. This confirms the suitability of the primmorph cell culture as a model sponge system. We also discovered mass mortality of green symbionts (Chlorophyta) was associated with a shift in the microbial communities of sponges/primmorphs. Microbes in diseased sponges, and infected primmorphs, belonged mainly to the phyla Bacteroidetes and Proteobacteria and these families Flavobacteriaceae, Burkholderiaceae, and Moraxellaceae. Primmorphs cell culture may provide a model to study interactions between these bacteria and their host and elucidate the cause of mass mortality events.


2009 ◽  
Vol 160 (1-2) ◽  
pp. 171-174 ◽  
Author(s):  
Dana Čerňanská ◽  
Barbara Paoletti ◽  
Ivica Kráľová-Hromadová ◽  
Raffaella Iorio ◽  
Patrícia Čudeková ◽  
...  

Author(s):  
A. Berezenko ◽  
◽  
V. Nedosekov ◽  
O. Godovskiy ◽  
◽  
...  

One of the most common viruses in the world that causes disease in cattle is the bovine coronavirus (BCoV). This virus is the causative agent of respiratory and gastrointestinal infections in newborn calves, resulting in significant economic losses in both dairy and meat farming. Considering the complex epizootic situation with the coronaviruses in the world and partial antigenic affinity of BCoV with coronaviruses of other species of animals and humans, the isolation of new strains of coronaviruses, their identification and optimization of cultivation conditions becomes extremely important and relevant. The aim of our research was to determine the features of methods of isolation of bovine coronavirus and to select methods for its cultivation in cell culture in order to obtain the virus with the highest titers of infectious activity. Isolation of BCoV was performed in monolayers of MDBK and the primary-trypsinized calf kidney culture cells, using 20 samples collected from calves with clinical signs of respiratory or/and gastrointestinal disease. 16 samples were positive for BCoV by means of Real-Time PCR test. Up to fifth serial passage, only 4 of these isolates presented typical syncytial cytopathic effect. It has been experimentally established that the continious calf kidney cell culture line (MDBK) and the primary-trypsinized calf kidney culture (CK) are suitable for BCoV isolation and accumulation. The infectious titer of bovine coronavirus at the level of the fifth passage in the cultures of MDBK and CK cells reached 5.54 ± 0.16 lg TCD50/ml and 5.59 ± 0.14 lg TCD50/ml, respectively. However, due to the high cost of obtaining primary-trypsinized cell cultures, this isolation method may be unacceptable to most pharmaceutical companies and laboratories. Also after 5 serial passages, the viral material was again examined in Real-Time PCR to confirm the isolation of BCoV - the study of 4 samples with a characteristic syncytial CPE had a positive result in Real-Time PCR. However, of the Real-Time PCR-positive 12 samples, the virus could not be isolated in continuous cell cultures of MDBK and Vero, as well as in primary-trypsinized cattle lung and kidney cell cultures. This fact may indicate the presence of different strains of BCoV circulation in farms in our country. Further research is planned to be focused on optimizing the methods and modes of BCoV strains isolation, as well as to identify and study the cultural properties of new strains of BCoV circulating in Ukraine. We will also continue the study of the obtained viral isolate for the subsequent development of tools for the diagnosis and immunoprophylaxis of coronavirus infection in veterinary medicine.


2020 ◽  
Vol 36 (6) ◽  
pp. 35-48
Author(s):  
D.V. Коchkin ◽  
G.I. Sobolkovа ◽  
А.А. Fоmеnkov ◽  
R.А. Sidorov ◽  
А.М. Nоsоv

The physiological characteristics of the callus cell cultures of Alhagi persarum Boiss et Buhse, a member of the legume family, widely used in folk medicine, have been studied. It was shown that the source of the explant was an important factor in the initiation of callusogenesis: more intense callusogenesis (almost 100%) was observed for explants from various organs of sterile seedlings, rather than intact plants (less than 30%). As a result, more than 20 lines of morphologically different callus cell cultures were obtained, and the growth parameters for the 5 most intensively growing lines were determined. The composition of fatty acids (FA) of total lipids and secondary metabolites in the most physiologically stable callus line Aр-207 was analyzed. Using capillary gas-liquid chromatography with mass spectrometric detection (GLC-MS), 19 individual C12--C24 FAs were identified, the main fraction of which were palmitic (~ 23%), stearic (~ 22%), linoleic (~ 14%) and α-linolenic (~ 33%) acids. The established atypical ratio of FAs (a simultaneous high content of both saturated FAs and polyunsaturated α-linolenic acid) is possibly due to the adaptation of cells to in vitro growth conditions. Phytochemical analysis of the secondary metabolites was carried out using ultra-performance liquid chromatography with electrospray ionization mass spectrometric detection (UPLC MS). Compounds belonging to different structural groups of isoflavones were found. Aglycones (calycosin, formononetin and afrormosin isomer), glucosides (formononetin glucoside), as well as esters of glucosides (malonylglycosides of calicosin, formononetin, afrormosin isomers, glycitein and genistein) were detected. These secondary metabolites are widespread in plants of the Fabaceae family; however, isoflavones are rare in representatives of the Alhagi genus. The presence of malonylated isoflavone glycosides in Alhagi spp. was shown for the first time. endemic plant species, Alhagi, in vitro cell culture, callus cell culture, isoflavones, fatty acids All studies were carried out using the equipment of the "Experimental Biotechnological Facility" and the "All-Russian Collection of Cell Cultures of Higher Plants" of IРР RAS. This work was supported by the Russian Foundation for Basic Research (RFBR), contract no.18-54-06021 (Az_a), and the Government of the Russian Federation, Megagrant Project no. 075-15-2019-1882.


2003 ◽  
Vol 69 (11) ◽  
pp. 6380-6385 ◽  
Author(s):  
R. Temmerman ◽  
L. Masco ◽  
T. Vanhoutte ◽  
G. Huys ◽  
J. Swings

ABSTRACT The taxonomic characterization of a bacterial community is difficult to combine with the monitoring of its temporal changes. None of the currently available identification techniques are able to visualize a “complete” community, whereas techniques designed for analyzing bacterial ecosystems generally display limited or labor-intensive identification potential. This paper describes the optimization and validation of a nested-PCR-denaturing gradient gel electrophoresis (DGGE) approach for the species-specific analysis of bifidobacterial communities from any ecosystem. The method comprises a Bifidobacterium-specific PCR step, followed by purification of the amplicons that serve as template DNA in a second PCR step that amplifies the V3 and V6-V8 regions of the 16S rRNA gene. A mix of both amplicons is analyzed on a DGGE gel, after which the band positions are compared with a previously constructed database of reference strains. The method was validated through the analysis of four artificial mixtures, mimicking the possible bifidobacterial microbiota of the human and chicken intestine, a rumen, and the environment, and of two fecal samples. Except for the species Bifidobacterium coryneforme and B. indicum, all currently known bifidobacteria originating from various ecosystems can be identified in a highly reproducible manner. Because no further cloning and sequencing of the DGGE bands is necessary, this nested-PCR-DGGE technique can be completed within a 24-h span, allowing the species-specific monitoring of temporal changes in the bifidobacterial community.


Nematology ◽  
2009 ◽  
Vol 11 (6) ◽  
pp. 847-857 ◽  
Author(s):  
Lieven Waeyenberge ◽  
Nicole Viaene ◽  
Maurice Moens

Abstract ITS1, the 5.8S rRNA gene and ITS2 of the rDNA region were sequenced from 20 different Pratylenchus species. Additionally, the same region was sequenced from seven populations of P. penetrans. After purifying, cloning and sequencing the PCR products, all sequences were aligned in order to find unique sites suitable for the design of species-specific primers for P. penetrans. Since ITS regions showed variability between and even within populations of P. penetrans, only three small DNA sequences were suitable for the construction of three potentially useful species-specific primers. New species-specific primers were paired with existing universal ITS primers and tested in all possible primer combinations. The best performing primer set, supplemented with a universal 28S rDNA primer set that served as an internal control, was tested in duplex PCR. The ideal annealing temperature, Mg2+ concentration and primer ratios were then determined for the most promising primer set. The optimised duplex PCR was subsequently tested on a wide range of different Pratylenchus spp. and 25 P. penetrans populations originating from all over the world. To test the sensitivity, the duplex PCR was conducted on DNA extracted from a single P. penetrans nematode mixed with varying amounts of nematodes belonging to another Pratylenchus species. Results showed that a reliable and sensitive P. penetrans species-specific duplex PCR was constructed.


1998 ◽  
Vol 10 (1) ◽  
pp. 3-10 ◽  
Author(s):  
G. M. Allan ◽  
F. McNeilly ◽  
S. Kennedy ◽  
B. Daft ◽  
J. A. Ellis ◽  
...  

Samples of lung, liver, kidney, pancreas, spleen, and lymph node from pigs with postweaning multisystemic wasting syndrome from California (USA) and samples of mesenteric lymph nodes from similarly diseased pigs from Brittany (France) were examined by light microscopy, in situ hybridization (ISH), and/or virus isolation. Whole genomic probes for porcine circovirus (PCV) and chicken anemia virus (CAV) were used for ISH. Tissue homogenate supernatants were inoculated onto PK/15 cells for virus isolation, and the presence of viral antigen and viral particles was verified by indirect immunofluorescence, ISH, and electron microscopy. Histologic examination of lung from pigs from California revealed interstitial pneumonia, alveolar epithelial hyperplasia, and basophilic nuclear and cytoplasmic inclusions in mononuclear cell infiltrates and various pulmonary epithelial cells. Granulomatous lymphadenitis with syncytial cells typified the lesions seen in the pigs from France. PCV-like nucleic acid was detected by ISH in lung, pancreas, lymph node, kidney, and liver in pigs from California. Positive signal was also obtained in lymph node sections from pigs from France. Probes for CAV were consistently negative. PK/15 cell cultures inoculated with lung preparations from diseased California pigs and mesenteric lymph node preparations from pigs from France had positive fluorescence by indirect staining for PCV using pooled polyclonal pig sera and hyperimmune rabbit serum and had variable staining with a panel of 7 monoclonal antibodies specific for cell culture contaminant PCV. PCV-like nucleic acid was also detected by ISH in cell cultures. Cytopathic effect was not observed Electron microscopic examination of inoculated cell cultures revealed 17-nm viral particles morphologically consistent with PCV No other virus particles were observed. Although genomic analysis for the definitive identification of these viral isolates remains to be done, the evidence provided strongly suggests that these tissue isolates are closely related to, although antigenically distinct from, the original PCV cell culture contaminant.


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