Cesar Alejandro Rosales Rodriguez

European 1: a globally important clonal complex of Mycobacterium bovis.

We have identified a globally important clonal complex of Mycobacterium bovis by deletion analysis of over one thousand strains from over 30 countries. We initially show that over 99% of the strains of M. bovis, the cause of bovine tuberculosis, isolated from cattle in the Republic of Ireland and the UK are closely related and are members of a single clonal complex marked by the deletion of chromosomal region RDEu1 and we named this clonal complex European 1 (Eu1). Eu1 strains were present at less than 14% of French, Portuguese and Spanish isolates of M. bovis but are rare in other mainland European countries and Iran. However, strains of the Eu1 clonal complex were found at high frequency in former trading partners of the UK (USA, South Africa, New Zealand, Australia and Canada). The Americas, with the exception of Brazil, are dominated by the Eu1 clonal complex which was at high frequency in Argentina, Chile, Ecuador and Mexico as well as North America. Eu1 was rare or absent in the African countries surveyed except South Africa. A small sample of strains from Taiwan were non-Eu1 but, surprisingly, isolates from Korea and Kazakhstan were members of the Eu1 clonal complex. The simplest explanation for much of the current distribution of the Eu1 clonal complex is that it was spread in infected cattle, such as Herefords, from the UK to former trading partners, although there is evidence of secondary dispersion since. This is the first identification of a globally dispersed clonal complex M. bovis and indicates that much of the current global distribution of this important veterinary pathogen has resulted from relatively recent International trade in cattle.

Understanding the relationship between Mycobacterium bovis spoligotypes from cattle in Latin American countries.

Spoligotyping is the most frequently used method for genotyping isolates of Mycobacterium bovis worldwide. In the current work, we compared spoligotypes from 1684 M. bovis isolates from Argentina (816), Brazil (412), Chile (66), Mexico (274) and Venezuela (116), obtained from cattle, humans, pigs, wild boars, farmed deer, goats, buffaloes, cats, and wild animals. A total of 269 different spoligotypes were found: 142 (8.4%) isolates presented orphan spoligotypes, whereas 1542 (91.6%) formed 113 different clusters. In cattle, SB0140 was the most representative spoligotype with 355 (24.6%) isolates, followed by SB0121 with 149 (10.3%) isolates. Clustering of spoligotypes ranged from 95.2% in Argentina to 85.3% in Mexico. Orphan spoligotypes were also variable, ranging from 23.7% in Mexico to 4.1% in Brazil. A large proportion of spoligotypes were common to the neighboring countries Argentina, Brazil and Chile. In conclusion, despite the diversity of spoligotypes found in the five countries studied, there are major patterns that predominate in these neighboring countries. These clusters may reflect a long-lasting active transmission of bovine tuberculosis or common historical origins of infection.

Comparison of three decontamination methods for Mycobacterium bovis isolation

Sixty samples of tissue fragments with lesions suggestive of tuberculosis from bovine abattoirs, kept in saturated solution of sodium borate, were subjected to four treatments: 4% NaOH (Petroff Method), 12 % H2SO4 and 1.5% HPC (1-Hexadecylpyridinium Chloride) decontamination, and physiological saline solution (control). The HPC method showed the lowest contamination rate (3%) when compared to control (88%, p<0.001), NaOH (33%, p<0.001) and H2SO4 (21.7%, p<0.002). Regarding the isolation success, the HPC method was better (40%) than the control (3%, p<0.001), NaOH (13%, p=0.001) and H2SO4 (1.7%, p<0.001) methods. These results indicate that HPC is an alternative to the Petroff method.

Evaluation of DNA extraction protocols for Brucella abortus pcr detection in aborted fetuses or calves born from cows experimentally infected with strain 2308

The objective of the present study was to improve the detection of B. abortus by PCR in organs of aborted fetuses from infected cows, an important mechanism to find infected herds on the eradication phase of the program. So, different DNA extraction protocols were compared, focusing the PCR detection of B. abortus in clinical samples collected from aborted fetuses or calves born from cows challenged with the 2308 B. abortus strain. Therefore, two gold standard groups were built based on classical bacteriology, formed from: 32 lungs (17 positives), 26 spleens (11 positives), 23 livers (8 positives) and 22 bronchial lymph nodes (7 positives). All samples were submitted to three DNA extraction protocols, followed by the same amplification process with the primers B4 and B5. From the accumulated results for organ, the proportion of positives for the lungs was higher than the livers (p=0.04) or bronchial lymph nodes (p=0.004) and equal to the spleens (p=0.18). From the accumulated results for DNA extraction protocol, the proportion of positives for the Boom protocol was bigger than the PK (p<0.0001) and GT (p=0.0004). There was no difference between the PK and GT protocols (p=0.5). Some positive samples from the classical bacteriology were negative to the PCR and viceversa. Therefore, the best strategy for B. abortus detection in the organs of aborted fetuses or calves born from infected cows is the use, in parallel, of isolation by classical bacteriology and the PCR, with the DNA extraction performed by the Boom protocol.

Isolation of rotavirus from asymptomatic dogs in Brazil

is subdivided both serologically, based on the inner capsid VP6 protein, and electropherotypically, based on the arrangement of the 11 dsRNA segments, into seven groups (A to G). Groups A, B, and C are found in both humans and animals, whereas groups D, E, F, and G have been found only in animals to date. In most cases, the electrophoretic pattern of the genome of group A rotaviruses is composed of four high-molecular-weight dsRNA segments (numbered 1 to 4), two middle-sized segments (5 and 6), a distinctive triplet of segments (7 to 9), and two smaller segments (10 and 11) (Estes and Kapikian, 2007). Canine rotavirus most often causes mild enteritis, especially in pups younger than two weeks, but the virus is also found in health animals (Schwers et al., 1983; Mochizuki et al., 1986). To date, only 10 isolates of canine rotavirus have been reported, including three isolates from the USA, two from France, two from Japan, two from Italy, and one from Korea (England and Poston, 1980; Fulton et al., 1981; Hoshino et al., 1982; Hoyois et al., 1982; Mochizuki and Hsuan,

Isolation of bovine coronavirus (BCoV) in monolayers of HmLu-1 cells

Isolation of BCoV was performed in monolayers of HmLu-1 cells, using 20 fecal samples from clinical cases of diarrhea in calves. Samples were positive for BCoV by means of hemagglutination / hemagglutination inhibition (HA/HI). Up to the fifth serial passage, 13 of these isolates presented syncytial cytopathic effect, similar to Kakegawa standard strain. When isolates were submitted to seroneutralization with gammaglobulin anti-bovine coronavirus, 8 of them were considered to be positive, once cytopathic effect was neutralized. After titration and seroneutralization in microplates, only three of them were confirmed as positive. The lineage HmLu-1 showed higher permissivity to BCoV isolation, but the low intensity of viral replication demonstrated that new methodologies should be developed for this purpose and then submitted to confirmation of isolation by means of seroneutralization.

Seroprevalence of antibodies against group A rotavirus in cattle from a pioneer frontier in Brazilian Amazon

O rotavirus e um agente etiologico de diarreias de distribuicao mundial, responsavel por grandes prejuizos economicos. Pesquisou-se a soroprevalencia de anticorpos anti-rotavirus do sorogrupo A em bovinos criados no municipio de Uruara, utilizando a tecnica da contraimunoeletroosmofore com a amostra NCDV como antigeno padrao. A prevalencia de propriedades positivas foi de 95.6-100%. Diferencas significativas foram observadas entre as sucessivas faixas etarias, quando a soropositividade aumentou dos grupos mais jovens para os mais velhos, bem como entre femeas e machos maiores de 1 ano, quando a soropositividade foi maior no primeiro grupo.

Comparison of methods for mycobacteria isolation from swine feces

Swine mycobacteriosis is an important cause of carcass condemnation at abattoirs. One of the best ways to recognize the etiologic agent involved, in live animals, is the fecal isolation, as 94% of the lesions are located in the digestive tract. Therefore, the goal of the present study was to compare the performance of four decontamination methods followed by inoculation in three different culture media, totalizing twelve procedures of mycobacteria search from swine fecal samples experimentally contaminated. The swine feces were artificially contaminated with 0.02 g of Mycobacterium avium, PIG-B strain, and subjected to mycobacteria isolation trial. The protocols used were: 1) modified Petroff or basic method; 2) modified Lowenstein-Jensen or acidic method; 3) modified Petroff or basic method with re-suspension in Amphotericin B; 4) modified Lowenstein-Jensen or acid method with re-suspension in Amphotericin B, followed by inoculation in Petragnani, Lowenstein-Jensen and Lowenstein-Jensen medium with antibiotics (Penicillin G and Nalidixic acid). There was a difference (p<0.05) between the mycobacterial recovery percentages from swine feces. The acid method with re-suspension in Amphotericin B solution and inoculation in Lowenstein-Jensen medium with antibiotics showed the best results (87% of mycobacteria recovery).

Evaluation of Four DNA Extraction Protocols for Brucella abortus Detection by PCR in Tissues from Experimentally Infected Cows with the 2308 Strain

This study compared 4 protocols for DNA extraction from homogenates of 6 different organs of cows infected with the Brucella abortus 2308 strain. The extraction protocols compared were as follows: GT (guanidine isothiocyanate lysis), Boom (GT lysis with the carrying suspension diatomaceous earth), PK (proteinase K lysis), and Santos (lysis by boiling and freezing with liquid nitrogen). Positive and negative gold standard reference groups were generated by classical bacteriological methods. All samples were processed with the 4 DNA extraction protocols and amplified with the B4 and B5 primers. The number of positive samples in the placental cotyledons was higher than that in the other organs. The cumulated results showed that the Santos protocol was more sensitive than the Boom (p=0.003) and GT (p=0.0506) methods and was similar to the PK method (p=0.2969). All of the DNA extraction protocols resulted in false-negative results for PCR. In conclusion, despite the disadvantages of classical bacteriological methods, the best approach for direct diagnosis of B. abortus in organs of infected cows includes the isolation associated with PCR of DNA extracted from the cotyledon by the Santos or PK methods.