Transmissible gastroenteritis (TGE) is a highly contagious enteric disease of pigs caused by an enteropathogenic coronavirus, TGEV. The sequence of the 5' end of the spike glycoprotein (S) gene best distinguishes this virus from related coronavirus. Since February 2003, a large number of unexplained outbreaks of gastroenteritis have occurred on pig farms in Havana. The problem was identified as TGE for the first time in May 2003. This paper describes the virological and molecular studies that led to this diagnosis. The disease was experimentally reproduced in susceptible piglets, a sow, and in weaned pigs. TGEV recovered from the diarrheic faeces of the sick piglets was identified by electron microscope negative staining, viral isolation in secondary swine kidney cultures, and by RT-PCR using previously reported primers. The PCR product amplified from the 5' end of the S gene was directly sequenced. The nucleotide sequence and the deduced amino acid residues of the amplified region are reported. A phylogenetic analysis was performed by comparing a 393-414 nucleotide stretch near the 5' end of the S gene in 36 viruses from different countries and with different isolation dates. The virus causing the outbreak in Cuba seems to be closely related to a TGEV previously isolated in the US Midwest. The source of infection remains unknown.Additional key words: coronavirus, phylogenetic analysis, RT-PCR, spike protein, TGEV. ResumenGastroenteritis transmisible en Cuba: reproducción experimental de la enfermedad y caracterización molecular del virus La gastroenteritis transmisible (TGE) es una enfermedad entérica y altamente contagiosa de los cerdos causada por un coronavirus enteropatogénico, el TGEV. El extremo 5' del gen de la glicoproteina S de las espículas de la envoltura, difiere en gran medida de otros coronavirus relacionados. A partir de febrero del 2003, se produjeron numerosos brotes de gastroenteritis en granjas de cría de cerdos de la provincia de La Habana; la enfermedad fue caracterizada como TGE y reportada por primera vez en mayo del 2003. Se presentan los resultados de los estudios virológicos y moleculares que condujeron a este diagnóstico. La enfermedad fue reproducida experimentalmente en cerditos susceptibles, una cerda y su camada. El TGEV fue aislado de heces diarreicas provenientes de cerditos enfermos recién nacidos; se confirmó su identidad por tinción negativa al microscopio electrónico, aislamiento viral en cultivo primario de riñón de cerdo y RT-PCR usando cebadores específicos previamente descritos. El producto de PCR amplificado a partir del extremo 5' del gen de la proteína S fue secuenciado directamente. Se reporta la secuencia nucleotídica y la de aminoácidos deducida de la región amplificada. El análisis filogenético comparando las secuencias de un fragmento de 393-414 nucleótidos del extremo 5' del gen S de 36 virus de diferentes países y fechas de aislamiento, muestra que el virus que causó los brotes en Cuba parece estar muy relacionado con un aislado del medio oeste de EEUU,...
Bovine coronavirus (BCoV) infection causes epidemics of acute diarrhea in calves and winter dysentery (WD) in adult cattle. The disease in adult cattle can cause a decrease in milk production resulting in serious economic losses. In Cuba, BCoV infections have not been previously reported. During 2004, many outbreaks of enteric disease have occurred in adult cattle from thirteen dairy farms in the central and western part of the island. The clinical features of the outbreaks resembled those of WD, such as decrease in milk production and, 24 hours later, diarrhea, sometimes bloody, that lasted until the animals recovered in about 7-15 days. Laboratory confirmation of BCoV infection was provided by hemagglutination test (HAT) in 24 samples from four dairy farms from three provinces; RT-PCR assays confirmed the presence of BCoV in three of these samples. Cell culture isolation in secondary calf kidney cells was obtained from four pools of fecal diarrheic samples from each dairy farm. It was remarkable that the disease was also observed during summer. Studies of molecular characterization of the viral strain are in progress.
This research aimed to assess the biofilm formation ability of Campylobacter strains under temperature and oxygen stress conditions, similar to those found in the industrial environment, to explain the persistence of this pathogen on the poultry slaughter line. A collection of C. jejuni and C. coli isolates (n = 143) obtained from poultry samples (cecal content and neck skin), collected at slaughterhouse level, from diverse flocks, on different working days, was genotyped by flaA -restriction fragment length polymorphism ( RFLP ) typing method. A clustering analysis resulted in the assignment of 10 main clusters, from which 15 strains with different flaA -RFLP genotypes were selected for the assessment of biofilm formation ability and antimicrobial susceptibility. Biofilm assays, performed by crystal violet staining method, were conducted with the goal of mimicking some conditions present at the slaughterhouse environment, based on temperature, atmosphere, and contamination levels. Results indicated that many C. jejuni strains with similar flaA -RFLP profiles were present at the slaughterhouse on different processing days. All the strains tested (n = 15) were multidrug-resistant except for one. Biofilm formation ability was strain-dependent, and it appeared to have been affected by inoculum concentration, temperature, and tolerance to oxygen levels. At 10°C, adherence levels were significantly lower than at 42°C. Under microaerobic and aerobic atmospheres, at 42°C, 3 strains ( C. jejuni 46E, C. jejuni 61C, and C. coli 65B) stood out, exhibiting significant levels of biofilm formation. C. jejuni strains 46E and 61C were inserted in clusters with evidence of persistence at the slaughterhouse for a long period of time. This study demonstrated that Campylobacter strains from broilers are capable of forming biofilms under conditions resembling the slaughterhouse environment. These results should be seen as a cue to improve the programs of hygiene implemented, particularly in those zones that can promote biofilm formation.
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