Isabel Azevedo Carvalho

Escherichia coli from clinical mastitis serotypes and virulence factors

In the current study, the virulence factors in Escherichia coli isolates from bovine mastitis were investigated, and the connection between these factors and infection was evaluated using phenotypic and genotypic analyses. Twenty-seven E. coli isolates were analyzed, and 2 were shown to produce verotoxin. All isolates had the ability to produce biofilms, although at different levels. One isolate was found to be sensitive to the bactericidal activity of bovine serum, 11 were intermediate, and 15 were resistant. Some isolates showed resistance to trimethoprim sulfa (9) and ampicillin (4), intermediate resistance to neomycin (1) and trimethoprim sulfa (5), and simultaneous resistance to ampicillin and trimethoprim sulfa (4). The fimH gene was found in all isolates and was associated with other virulence markers: pap (1), stb (8), cs31a (3), stb and vt2 (2), cs31a and stb (3), east1 and kps (1), stb and east1 (1), cs31a and east1 (1), and cs31a, stb, pap, and iucD (1). Serogroups were determined for 3 isolates: O93:H4, O83:H19, and O15:H11. Phylogenetic analysis showed that 23 isolates belonged to group A and 4 belonged to B1. The findings revealed that these E. coli isolates are opportunistic pathogens with different virulence factors. The results indicate that the pathogenicity route of E. coli in bovine mastitis is not a consequence of 1 specific virulence factor.

Short communication: Recovery of viable Mycobacterium avium subspecies paratuberculosis from retail pasteurized whole milk in Brazil

Mycobacterium avium ssp. paratuberculosis (MAP) is the etiological agent of paratuberculosis, a chronic granulomatous enteritis that affects all ruminants worldwide. Some researchers have indicated a possible role of MAP in Crohns disease. Despite extensive research and large and important advances in the past few decades, the etiology of Crohns disease remains indefinite. The most probable transmission route of MAP from animals to humans is milk and dairy products. Mycobacterium avium ssp. paratuberculosis has already been detected in milk samples worldwide, and some studies have reported that MAP is resistant to pasteurization. In Brazil, MAP has been reported in raw milk samples; however, Brazilian retail pasteurized milk has not yet been tested for viable MAP. The aim of this study was to investigate MAP in pasteurized milk in the region of Viçosa (Minas Gerais, Brazil). Thirty-seven samples were collected and processed for culture of MAP. One colony similar to MAP was observed and confirmed by IS900-nested PCR and sequencing. Analysis revealed 97 to 99% identity with the MAP K-10 strain. This study is the first report of the presence of MAP in retail pasteurized whole milk in Brazil.

Short communication: Detection of Mycobacterium avium subspecies paratuberculosis by polymerase chain reaction in bovine milk in Brazil

Mycobacterium avium ssp. paratuberculosis (MAP) is the causative agent of paratuberculosis, or Johnes disease, a chronic granulomatous enteritis that affects all ruminants worldwide. Since the isolation of MAP from intestinal tissue of human patients bearing Crohns disease, there has been a debate on the possibility of this agent playing a role in the etiology of Crohns disease. Milk could be the potential vehicle for transmission to humans. Mycobacterium avium ssp. paratuberculosis has already been detected in milk samples worldwide. In Brazil, detection of MAP is uncommon; however, it has already been detected by bacterial isolation and serological test. The aim of this study was to investigate the presence of MAP, by PCR, in raw milk samples in the region of Viçosa, Minas Gerais State, Brazil. Of 222 milk samples evaluated, 8 (3.6%) quarter milk samples amplified fragments of similar size to that expected of 626 bp. These fragments were cloned and sequenced. The genetic analysis revealed a 99% identity match between the sequences obtained in this study and the insertion sequence IS900 deposited in the GenBank. In the analyzed milk samples, MAP DNA was detected, confirming its presence in dairy cattle in the region of Viçosa. This is the first report of MAP presence in raw milk samples in Brazil.

Short communication: Viable Mycobacterium avium subspecies paratuberculosis in retail artisanal Coalho cheese from Northeastern Brazil.

Mycobacterium avium ssp. paratuberculosis (MAP) is the etiologic agent of paratuberculosis and it potentially plays a role in Crohns disease. In humans, the main route of transmission of MAP might be the intake of contaminated milk and dairy products. Considering that MAP has already been detected in many types of cheese in different counties, and that Coalho cheese is an important dairy product in northeastern Brazil, the aim of this study was to report the first detection of MAP in retail Coalho cheese in Brazil by PCR and culture. Of 30 retail Coalho cheese samples, 3 (10%) amplified fragments of a similar size to that expected (626 bp) were obtained and viable MAP was recovered by culture from 1 (3.3%) sample. The DNA from the positive culture sample was sequenced and showed 99% identity with the insertion sequence IS900 deposited in GenBank. It was possible to identify the presence of MAP-specific DNA in the analyzed samples for the first time in Brazil, and to recover viable cells from retail Coalho cheese.

Genetic evaluation of IS900 partial sequence of Mycobacterium avium subsp. paratuberculosis Brazilian isolates from bovine milk

Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of paratuberculosis. Insertion sequence IS900 is used for the identification of MAP. The objective of this study was to verify the genetic conservation of IS900 sequences in raw milk samples. To evaluate genetic conservation, 206 quarter milk samples and 16 bulk-tank milk samples were collected. DNA extraction and IS900 PCR were performed in all samples. Six samples amplified the expected fragment. To confirm the identity of the amplified fragments, PCR products were cloned and sequenced. The resulting sequences were compared with other MAP sequences from GenBank, and it was possible to identify eight polymorphic regions and to form five distinct haplotypes. The number of mutations in each haplotype was verified. IS900 sequence is a very well-conserved sequence that could be used as tool for the molecular detection of this agent and epidemiological purposes. The results showed the first genetic analysis on Brazilian isolates of MAP.

Diagnosis of paratuberculosis in cattle: microbiological culture, serology and PCR

The aim of this study was to confirm clinical diagnosis of paratuberculosis in two cows showing suggestive clinical signs of the disease. Based on clinical signs, in culture and in IS900 PCR results from the individual milk samples it was possible to diagnose paratuberculosis in the cows studied.

Mycobacterium avium ssp. paratuberculosis vs Crohn’s Disease

Crohns disease (CD) is a chronic inflammatory bowel disease, with the potential to affect any segment of the gastrointestinal tract. Despite the great advances in recent decades, which provided a better understanding of the pathogenesis of the disease, it is yet to be completely elucidated. Presence of genetic factors, luminal factors such as microflora and factors related to the intestinal barrier and immunoregulation are pieces that interact with each other and with environmental factors. The possibility of an infectious etiology has always been widely discussed. In this context, Mycobacterium avium subspecies paratuberculosis (MAP) has attracted the interest of many researchers because of the similarity between paratuberculosis and CD. In 1913, two decades before the description of CD in 1932, T. K. Dalziel made associations between chronic cases of enteritis in humans and paratuberculosis in cattle (Behr and Kapur, 2008). Some genetic studies also support the role of MAP in CD and susceptibility genes have been identified, which encode proteins involved in the innate immunity defense against intracellular bacteria. However, no study is conclusive about a causal relationship. It is not possible to conclude that a single agent is solely responsible for the etiology of CD: a multifactorial cause is much more likely (Grant et al., 2001). Whereas the causal relationship has not been established, therapeutic implications require further studies. Results of these studies could help answer questions about the role of MAP in the etiology of CD.

Paratuberculose em pequenos ruminantes domésticos

Mycobacterium avium subesp. paratuberculosis (MAP) e o agente etiologico da paratuberculose em ruminantes domesticos e silvestres. Em caprinos e ovinos causa uma enterite granulomatosa cronica com emagrecimento progressivo seguido de morte, raramente a diarreia e observada. Nesse aspecto, a doenca e considerada uma ameaca mundial aos rebanhos, pois pode permanecer no estadio subclinico por anos, manifestando perdas indiretas na producao animal e na disseminacao do agente. Alem disso, existem relatos da possivel relacao de MAP com a doenca de Crohn, determinando, assim, a sua relevância na saude publica. O Brasil nao dispoe de dados quantificando as reais perdas produtivas nos rebanhos acometidos pela doenca, e poucas informacoes do comprometimento de caprinos e ovinos no pais sao relatadas. Assim, este artigo busca revisar a paratuberculose em pequenos ruminantes domesticos.

Paratuberculosis in Latin America: a systematic review

Latin America is the definition of the American group, where languages of Latin origin are spoken, including countries in South, Central, and North America. Paratuberculosis is a gastrointestinal contagious chronic disease that affects ruminants, whose etiological agent is the bacilli Mycobacterium avium subsp. paratuberculosis (MAP). Paratuberculosis is characterized by intermittent diarrhea, decreased milk production, dehydration, and progressive weight loss and is possibly involved in Crohn’s disease, a human intestinal disease. MAP is resistant to environmental factors, pasteurization, and water disinfection, which coupled with the subclinical-clinical nature of the disease, and makes paratuberculosis a relevant socioeconomic and public health issue, justifying the descriptive review of research on the disease carried out in Latin American countries. A survey of articles, published until September 2016, on the Scopus database, PubMed, Agris, and Science Direct, about detection of the agent and the disease in Latin America, without restrictions to the date of the research was performed. The keywords were as follows: “paratuberculosis,” “Mycobacterium avium subsp. paratuberculosis,” “cattle,” “milk,” “wildlife,” “goat,” “ovine,” “dairy,” and the name of each country in English. Studies found from nine of the 20 Latin America countries, 31 related to Brazil, 17 to Argentina, 14 to Chile, eight to Colombia, six to Mexico, two to Peru, two to Venezuela, and one to Panama and to Bolivia, each. The agent was detected in cattle, goats, sheep, domesticated water buffalo, and wild animals. Microbiological culture, PCR, and ELISA were the frequent techniques. The small number of studies may result in overestimation or underestimation of the real scenario.

Presence of Mycobacterium avium subsp. paratuberculosis (MAP) in Brazilian patients with inflammatory bowel diseases and in controls

CONTEXT AND OBJECTIVE Mycobacterium avium subsp. paratuberculosis (MAP) has attracted the interest of researchers because of similarities between paratuberculosis and Crohns disease (CD). The aim of this study was to evaluate the frequency of MAP through cultures, histology and polymerase chain reaction (PCR) on intestinal biopsies from Brazilian CD patients. Quantitative real time PCR (qRT-PCR) was performed on positive samples. DESIGN AND SETTING Analytical cross-sectional study with control group at two federal universities. METHODS Fresh samples were collected from 25 patients; five with CD, eight with ulcerative colitis (UC) and 12 controls with non-inflammatory bowel disease (nIBD). Formalin-fixed paraffin-embedded (FFPE) samples from 143 patients were also collected: 44 CD, 49 UC and 56 nIBD. RESULTS None of the fresh samples was positive for MAP. Five FFPE samples (one CD, two UC and two nIBD) and three fresh samples (one in each group) were positive through IS900-PCR. qRT-PCR was performed on these eight samples. Among the FFPE samples, there were 192.12 copies/μl in the CD group, 72.28 copies/μl in UC and 81.43 copies/μl in nIBD. Among the fresh samples, there were 432.99 copies/μl, 167.92 copies/μl and 249.73 copies/μl in the CD, UC and nIBD groups, respectively. The highest bacterial load was in the CD group. CONCLUSION This study does not provide evidence for a role of MAP in the etiology of CD, although MAP DNA was detected in all three patient groups. This is the first report of MAP presence in human intestinal biopsies in Brazil.