Sperm viability, serological, molecular, and modified seminal plasma agglutination tests in the diagnosis of Leptospira in the semen and serum of bovine bulls.

The present study investigated the serum microscopic agglutination test (MAT) among 203 bovine bulls with reproduction by natural means, without apparent signs of orchitis or inflammation of accessory reproductive glands. Simultaneously, the semen of all bulls was subjected to sperm viability analysis and PCR based on the 16S rRNA gene. PCR-positive results of semen samples were confirmed by sequencing. A modified seminal plasma agglutination (MSPA) test, replacing the blood serum of all bulls in the MAT with seminal plasma was performed as well. Eight (8/203 = 3.9%) semen samples from bulls were considered nonviable (necrospermia and azoospermia) without relation to the PCR diagnosis. No agglutinin titers were identified in MSPA test. A high frequency (132/203 = 65%) of leptospiral agglutinin titers was identified in the MAT, particularly for the Sejroe serogroup (Hardjo CTG, 100/203 = 49.3%; Wolffi 74/203 = 36.4%; Guaricura 72/203 = 35.5%; and Hardjoprajitno 56/203 = 27.6%). Three (3/203 = 1.5%) semen samples of bulls were positive in the PCR, but these results were not confirmed by sequencing. The high frequency of serovars from the Sejroe serogroup typically adapted to bovines indicates the need for measures for the prophylaxis/control of the pathogen on the sampled farms. Discrepancies among the MAT, sperm viability, and molecular detection of leptospires in semen highlight the need for a combination of methods to diagnose leptospirosis in bovine bulls. To our knowledge, modified seminal plasma agglutination is described for the first time here to investigate anti-Leptospira antibodies produced locally in the genital tract in the diagnosis of bovine leptospirosis among bulls that reproduce by natural means.

Adherence ability and serum resistance of different hospital clusters of Acinetobacter baumannii.

The role of mechanical ventilation and catheters in favouring Acinetobacter baumannii infections needs to be better understood. This study evaluated the adherence of 19 isolates of different hospital clusters of A. baumannii to abiotic surfaces and epithelial cells (HEp-2). Of the hydrophobic isolates, 80% adhered to polystyrene, indicating a close relationship between hydrophobicity and adherence. All isolates adhered to epithelial cells to different degrees, and 73·7% showed an aggregated pattern. Analysis of the serum resistance of catheter-tip isolates showed that all were resistant. These worrisome results showed that the high capacity of A. baumannii to adhere to surfaces and survive in human serum could hinder treatment and control of this pathogen.

Comparative study of IS711 and bcsp31-based polymerase chain reaction (PCR) for the diagnosis of human brucellosis in whole blood and serum samples.

BACKGROUND: Clinical diagnosis of human brucellosis (HB) is often difficult due to non-specific symptoms. Immunological tests have been the most common method used in HB diagnosis, but molecular methods based on quantitative polymerase chain reaction (qPCR) have largely replaced these diagnostic methods. The aim of this study was to validate a HB diagnostic qPCR method; assessing different target Brucella genes, and the influence of biological matrices (serum vs. whole blood) on analytical parameters. MATERIAL AND METHODS: Two target genes, IS711 and bcsp31, for HB molecular diagnosis were evaluated, together with biological matrix type (whole blood and serum) using samples spiked with Brucella abortus. In addition, diagnostic parameters of this qPCR method were evaluated in paired whole blood and serum samples from patients with suspected HB. RESULTS: Both genes could be potential diagnostic targets, but IS711 showed a lower limit of detection. In spiked matrix experiments, whole blood showed a lower limit of detection than serum after probit regression (224 vs. 3681 CFU/mL) and ANOVA analysis showed a significant (p < 0.001) difference between the Cq of whole blood at all dilutions and that of serum. In 12 paired clinical samples, no serum samples and only one whole blood sample tested positive for Brucella using this qPCR detection method. CONCLUSIONS: This standardized qPCR-based Brucella detection method could improve diagnosis of HB, serving as a rapid, highly sensitive, and specific test. Whole blood is better suited to qPCR-based HB diagnosis due to the presence of higher target DNA loads in this matrix, compared to serum.

Innate immunity in chicken lines developed by EMBRAPA Suínos e Aves: antimicrobial activity of macrophages and serum

The objective of this study was to compare the antimicrobial activity of macrophages and serum in laying hen (MM, CC, and CCc) and broiler chicken lineages (TT and LL). Macrophages were evaluated for phagocytic and antimicrobial activity. Microbicidal serum activity was evaluated by the resistance test for serum and the agar test. The results showed that phagocytic activity was higher in males of the MM strain, with 13% of macrophages presenting phagocytosis, while the other lineages studied, and even female MM, presented a rate of 6% of phagocytic cells. However, antimicrobial activity in macrophages from males of CCc lineage and females of TT lineage were higher, eliminating more than 30% of the Salmonella enterica inoculum, while in the other strains, the results were similar, with inoculum reduction below 30%. In the serum resistance assay, female laying lines presented higher antibacterial activity than female broiler lines. In the trials to evaluate the microbicide activity of the serum, females of both broiler and laying lineages presented higher performance when compared with males of the same lineage. Females of laying hen lines (MM and CC) present a greater antibacterium activity than males. These results can contribute to a better understanding of the immune response in broiler chicken and laying hen lineages, to aid development of lineages of birds more resistant to pathogens.

Analysis of irradiated Argentinean fetal bovine serum for adventitious agents.

Fetal bovine serum (FBS) used in cell culture may be contaminated with adventitious agents, which can affect the production of biologicals and the results of clinical laboratory tests. We carried out a retrospective study to determine the incidence of adventitious agent contamination of Argentinean irradiated FBS dating from 2015 to 2019. We analyzed FBS batches for mycoplasma and adventitious viruses (bovine pestiviruses, bovine adenovirus, bluetongue virus, bovine parainfluenza virus 3, rabies virus, bovine parvovirus, bovine herpesvirus 1, bovine respiratory syncytial virus, and reovirus). Cell passages followed by direct immunofluorescence were carried out to check viability of the mentioned adventitious agents. Also, molecular detection of mycoplasma and pestiviruses was performed on the FBS samples. The presence of neutralizing antibodies against pestiviruses was determined. Molecular analyses indicated that frequencies of mycoplasma and pestiviruses in FBS were 14% and 84%, respectively. All of the batches were seronegative for pestiviral antibodies. After cell passages, all FBS samples were negative for hemadsorbent agents and by immunofluorescence for all of the viral species analyzed; PCR assays were negative for mycoplasma and pestiviruses. Our results demonstrate that, of all adventitious agents tested, local FBS batches only had traces of mycoplasma and pestiviruses; gamma irradiation was effective in inactivating them.

Sporulation on blood serum increases the virulence of Mucor circinelloides.

Mucor circinelloides is an opportunistic human pathogen that is used to study mucormycosis, a rare but lethal infection in susceptible immunosuppressed patients. However, the virulence characteristics of this pathogen have not been fully elucidated. In this study, sporangiospores (spores) produced on YPG medium supplemented with native blood serum increased the virulence of M. circinelloides compared with spores produced on YPG supplemented with denatured blood serum or on YPG alone. The spores produced from YPG supplemented with native blood serum increased nematode death and led to significant increases in interleukin (IL)-6, IL-1ß, macrophage inhibitory protein-2, and tumour necrosis factor α mRNA levels in liver and lung tissues from infected diabetic mice compared with those in tissues from animals infected with spores produced in the presence of YPG supplemented with denatured blood serum or of YPG alone. Moreover, spores produced from cultures supplemented with native blood serum showed increased germination rates and longer hyphae compared with other spores. The spores produced in YPG supplemented with native blood serum also enhanced resistance to stress factors and H2O2 and increased thermotolerance compared with spores produced under other conditions. In addition, spores produced in presence of blood serum increased the ability of the pathogen to survive in the presence of macrophages. Taken together, our results showed that these factors were important features for fungal virulence in humans and suggested that thermolabile components in the blood serum may induce M. circinelloides virulence.

Perfil sérico proteico de vacas Holandesas no período de transição / Serum protein profile of Holstein cows during the transition period

O objetivo deste trabalho foi avaliar, de forma longitudinal, o perfil sérico proteico de 13 vacas Holandesas durante o período de transição. Amostras de sangue (n=78) foram coletadas semanalmente, da segunda semana pré-parto (M-2) até a terceira semana pós-parto (M3), para determinação do perfil sérico proteico, por meio de teste bioquímico (proteínas séricas totais - PT) e eletroforese em gel de poliacrilamida SDS-PAGE, para as outras proteínas analisadas. Os valores de PT diminuíram de forma gradativa (P=0,000) de M-2 (6,4g/dL) a M0 (6,2g/dL), aumentando nos momentos subsequentes (M3=7,3g/dL). As concentrações da IgG de cadeia pesada (M-2=919,4; M-1=1074,5mg/dL) e de cadeia leve (M-2=393,9; M-1=466,7mg/dL) foram menores no pré-parto em relação ao pós-parto (M1=1283,3; M2=1374,2 e M3=1630,3 mg/dL para IgG pesada e M1=463,4; M2=573,7; M3=651,8mg/dL para IgG leve). Para a IgA, houve diminuição nos valores (P=0,001), de M-2 (51,9mg/dL) a M1 (34,0mg/dL), e aumento em M2 (45,4g/dL) e M3 (62,6g/dL). Os valores de haptoglobina (Hp) e ceruloplasmina (Cp) aumentaram (P=0,000) de M-2 (Hp=16,6mg/dL; Cp=8,6mg/dL) a M3 (Hp=60,9mg/dL; Cp=127,1mg/dL). A albumina apresentou ligeiras variações durante o período de transição (P=0,000), enquanto a transferrina sérica (P=0,101) e a glicoproteína ácida (P=0,105) foram estáveis. O escore de condição corporal (ECC) também foi analisado durante o período de transição, verificando-se diferença (P=0,003) entre M-2 (ECC=4,0) e M1 (ECC=3,0). Foi relatada ainda a ocorrência de distocias (4/13), retenção de placenta (1/13) e hipocalcemia (1/13) no dia da parição (M0) e infecções uterinas (5/13) e cetose (1/13) ocorridas no pós-parto. Concluiu-se, portanto, que houve aumento nas concentrações séricas de Hp e Cp e diminuição nos valores de imunoglobulina e transferrina em vacas Holandesas no período de transição, relacionados às doenças ocorridas nesse período e ao elevado ECC, promovendo modificações metabólicas e imunossupressão.(AU)

The aim of this study was to evaluate longitudinally the serum protein profile of 13 Holstein cows during the transition period. Blood samples (n=78) were taken weekly, from the second week before parturition (M-2) to the third week after parturition (M3) for determination of serum protein profile by biochemical tests (total serum protein - PT) and SDS-PAGE electrophoresis, for the other proteins analyzed. PT values decreased gradually (P = 0.000) from M-2 (6.4g/dL) to M0 (6.2g/dL), increasing in subsequent moments (M3 = 7.3g/dL). The concentrations of the heavy chain (M-2=919.4; M-1=1074.5mg/dL) and the light chain of IgG (M-2=393.9; M-1=466.7mg/dL) were lower in pre-calving compared to post calving (M1= 1,283.3; M2=1,374.2 and M3=1,630.3 mg/dL for the heavy chain, and M1=463.4; M2=573.7 and M3=651,8 mg/dL for the light chain of IgG). For IgA there was a decrease in the values (P=0.001), from M-2 (51.9mg/dL) to M1 (34.0mg/dL), and increase in M2 (45,4g/dL) and M3 (62,6g/dL). The values of haptoglobin (Hp) and ceruloplasmin (Cp) increased (P=0,000) from M-2 (Hp=16.6mg/dL; Cp=8.6 mg/dL) to M3 (Hp=60.9mg/dL; Cp=127.1mg/dl). Albumin showed slight variations during the transition period (P=0.000), while the serum transferrin (P=0.101) and acid glycoprotein (P=0.105) were stable. Body condition score (BCS) was also analyzed during the transition period, checking the difference (P=0.003) between M-2 (ECC= 4.0) and M1 (ECC=3.0). It was also reported the occurrence of dystocia (4/13), retained placenta (1/13) and hypocalcemia (1/13) on the day of calving (M0) and uterine infections (5/13) and ketosis (1/13) occurring in the post calving. In conclusion, there was an increase in serum Hp and Cp, and a decrease in the immunoglobulins and transferrin amounts in Holstein cows during the transition period, related to diseases occurring during this period and the high BCS, promoting metabolic changes and immunosuppression.(AU)

Perfil sérico proteico de vacas Holandesas no período de transição / Serum protein profile of Holstein cows during the transition period

O objetivo deste trabalho foi avaliar, de forma longitudinal, o perfil sérico proteico de 13 vacas Holandesas durante o período de transição. Amostras de sangue (n=78) foram coletadas semanalmente, da segunda semana pré-parto (M-2) até a terceira semana pós-parto (M3), para determinação do perfil sérico proteico, por meio de teste bioquímico (proteínas séricas totais - PT) e eletroforese em gel de poliacrilamida SDS-PAGE, para as outras proteínas analisadas. Os valores de PT diminuíram de forma gradativa (P=0,000) de M-2 (6,4g/dL) a M0 (6,2g/dL), aumentando nos momentos subsequentes (M3=7,3g/dL). As concentrações da IgG de cadeia pesada (M-2=919,4; M-1=1074,5mg/dL) e de cadeia leve (M-2=393,9; M-1=466,7mg/dL) foram menores no pré-parto em relação ao pós-parto (M1=1283,3; M2=1374,2 e M3=1630,3 mg/dL para IgG pesada e M1=463,4; M2=573,7; M3=651,8mg/dL para IgG leve). Para a IgA, houve diminuição nos valores (P=0,001), de M-2 (51,9mg/dL) a M1 (34,0mg/dL), e aumento em M2 (45,4g/dL) e M3 (62,6g/dL). Os valores de haptoglobina (Hp) e ceruloplasmina (Cp) aumentaram (P=0,000) de M-2 (Hp=16,6mg/dL; Cp=8,6mg/dL) a M3 (Hp=60,9mg/dL; Cp=127,1mg/dL). A albumina apresentou ligeiras variações durante o período de transição (P=0,000), enquanto a transferrina sérica (P=0,101) e a glicoproteína ácida (P=0,105) foram estáveis. O escore de condição corporal (ECC) também foi analisado durante o período de transição, verificando-se diferença (P=0,003) entre M-2 (ECC=4,0) e M1 (ECC=3,0). Foi relatada ainda a ocorrência de distocias (4/13), retenção de placenta (1/13) e hipocalcemia (1/13) no dia da parição (M0) e infecções uterinas (5/13) e cetose (1/13) ocorridas no pós-parto. Concluiu-se, portanto, que houve aumento nas concentrações séricas de Hp e Cp e diminuição nos valores de imunoglobulina e transferrina em vacas Holandesas no período de transição, relacionados às doenças ocorridas nesse período e ao elevado ECC, promovendo modificações metabólicas e imunossupressão.(AU)

The aim of this study was to evaluate longitudinally the serum protein profile of 13 Holstein cows during the transition period. Blood samples (n=78) were taken weekly, from the second week before parturition (M-2) to the third week after parturition (M3) for determination of serum protein profile by biochemical tests (total serum protein - PT) and SDS-PAGE electrophoresis, for the other proteins analyzed. PT values decreased gradually (P = 0.000) from M-2 (6.4g/dL) to M0 (6.2g/dL), increasing in subsequent moments (M3 = 7.3g/dL). The concentrations of the heavy chain (M-2=919.4; M-1=1074.5mg/dL) and the light chain of IgG (M-2=393.9; M-1=466.7mg/dL) were lower in pre-calving compared to post calving (M1= 1,283.3; M2=1,374.2 and M3=1,630.3 mg/dL for the heavy chain, and M1=463.4; M2=573.7 and M3=651,8 mg/dL for the light chain of IgG). For IgA there was a decrease in the values (P=0.001), from M-2 (51.9mg/dL) to M1 (34.0mg/dL), and increase in M2 (45,4g/dL) and M3 (62,6g/dL). The values of haptoglobin (Hp) and ceruloplasmin (Cp) increased (P=0,000) from M-2 (Hp=16.6mg/dL; Cp=8.6 mg/dL) to M3 (Hp=60.9mg/dL; Cp=127.1mg/dl). Albumin showed slight variations during the transition period (P=0.000), while the serum transferrin (P=0.101) and acid glycoprotein (P=0.105) were stable. Body condition score (BCS) was also analyzed during the transition period, checking the difference (P=0.003) between M-2 (ECC= 4.0) and M1 (ECC=3.0). It was also reported the occurrence of dystocia (4/13), retained placenta (1/13) and hypocalcemia (1/13) on the day of calving (M0) and uterine infections (5/13) and ketosis (1/13) occurring in the post calving. In conclusion, there was an increase in serum Hp and Cp, and a decrease in the immunoglobulins and transferrin amounts in Holstein cows during the transition period, related to diseases occurring during this period and the high BCS, promoting metabolic changes and immunosuppression.(AU)

Molecular detection of Brucella species in patients suspicious of Brucellosis from Zanjan, Iran.

Brucella is an intracellular pathogen capable of infecting animals and humans. The aim of this study was to identify Brucella spp in sera of high risk individuals by a polymerase chain reaction (PCR)-based method. A total of 180 patients suspected to have Brucellosis were examined by serological tests. To establish a PCR protocol for diagnosis of active brucellosis, DNA was extracted from the serum samples by using a commercial kit. PCR amplification was done for detection of Brocella DNA using BCSP31 target gene and IS711 locus. The PCR assay showed that an amplicon of 223 bp was obtained in 73.8% (133/180) of the tested sera using primers (B4/B5) derived from a gene encoding the 31-kDa Brucella abortus antigen. In another PCR, an amplicon of 498 bp was obtained in 63.8% (115/180) of the samples using Brucella abortus-specific primers derived from a locus adjacent to the 3'-end of IS711, and also an amplicon of 731 bp was produced in 4.4% (8/180) of the tested samples using Brucella melitensis-specific primers. When the Wright method was used as a gold standard, the sensitivity and specificity of the PCR technique for genus identification were found to be 96 and 80.7%, respectively. However, the sensitivity value obtained with the species-specific PCR method was 82%, and specificity was similar to that previous reported. This is the first report of a high frequency of Brucella abortus in patients suspicious of Brucellosis from the Zanjan province.

Molecular detection of Brucella species in patients suspicious of Brucellosis from Zanjan, Iran

Brucella is an intracellular pathogen capable of infecting animals and humans. The aim of this study was to identify Brucella spp in sera of high risk individuals by a polymerase chain reaction (PCR)-based method. A total of 180 patients suspected to have Brucellosis were examined by serological tests. To establish a PCR protocol for diagnosis of active brucellosis, DNA was extracted from the serum samples by using a commercial kit. PCR amplification was done for detection of Brocella DNA using BCSP31 target gene and IS711 locus. The PCR assay showed that an amplicon of 223 bp was obtained in 73.8% (133/180) of the tested sera using primers (B4/B5) derived from a gene encoding the 31-kDa Brucella abortus antigen. In another PCR, an amplicon of 498 bp was obtained in 63.8% (115/180) of the samples using Brucella abortus-specific primers derived from a locus adjacent to the 3'-end of IS711, and also an amplicon of 731 bp was produced in 4.4% (8/180) of the tested samples using Brucella melitensis-specific primers. When the Wright method was used as a gold standard, the sensitivity and specificity of the PCR technique for genus identification were found to be 96 and 80.7%, respectively. However, the sensitivity value obtained with the species-specific PCR method was 82%, and specificity was similar to that previous reported. This is the first report of a high frequency of Brucella abortus in patients suspicious of Brucellosis from the Zanjan province.

Molecular detection of Brucella species in patients suspicious of Brucellosis from Zanjan, Iran

Brucella is an intracellular pathogen capable of infecting animals and humans. The aim of this study was to identify Brucella spp in sera of high risk individuals by a polymerase chain reaction (PCR)-based method. A total of 180 patients suspected to have Brucellosis were examined by serological tests. To establish a PCR protocol for diagnosis of active brucellosis, DNA was extracted from the serum samples by using a commercial kit. PCR amplification was done for detection of Brocella DNA using BCSP31 target gene and IS711 locus. The PCR assay showed that an amplicon of 223 bp was obtained in 73.8% (133/180) of the tested sera using primers (B4/B5) derived from a gene encoding the 31-kDa Brucella abortus antigen. In another PCR, an amplicon of 498 bp was obtained in 63.8% (115/180) of the samples using Brucella abortus-specific primers derived from a locus adjacent to the 3'-end of IS711, and also an amplicon of 731 bp was produced in 4.4% (8/180) of the tested samples using Brucella melitensis-specific primers. When the Wright method was used as a gold standard, the sensitivity and specificity of the PCR technique for genus identification were found to be 96 and 80.7%, respectively. However, the sensitivity value obtained with the species-specific PCR method was 82%, and specificity was similar to that previous reported. This is the first report of a high frequency of Brucella abortus in patients suspicious of Brucellosis from the Zanjan province.

Evaluation of nested polymerase chain reaction for the early detection of Leptospira spp. DNA in serum samples from patients with leptospirosis.

The aim of this study was to analyse the nested polymerase chain reaction (nested PCR) in human serum samples of patients with clinical manifestations of leptospirosis. The cases of leptospirosis were defined by the microagglutination test (MAT). The samples were collected in 2010. Of 1042 serum samples collected from 521 patients, 28 (5.4%) were considered positive cases of leptospirosis, and 493 (94.6%) were negative. Twenty-three confirmed cases had no MAT-detectable antibodies in the acute sample (mean of 5.6 days after onset). Nested PCR was positive in 22/23 (95.7%) patients during the acute phase of the disease, with negative results by MAT. Nested PCR was negative in all convalescent serum samples with positive results by MAT. All negative cases of leptospirosis were negative by nested PCR. The nested PCR is an alternative diagnostic tool for early detection of leptospires in sera during the first 7 days of the disease.

Serum samples can be substituted by plasma samples for the diagnosis of paratuberculosis.

Employing plasma samples rather than serum samples for serological paratuberculosis diagnosis is practical, especially when bovine TB is assessed in the same cattle herd with the gamma interferon bovine avian (IFN-γ BA) test. We demonstrate that antibody titers in serum and plasma samples, utilizing the PARACHECK(®) ELISA kit, are highly comparable (Cohen's kappa test, k=0.955). We conclude that serum can be replaced with plasma in this commercially available antibody detection assay resulting in working hour savings for sampling and blood sample work-up and cost reductions for materials and sample storage.

Application of PCR in serum samples for diagnosis of paracoccidioidomycosis in the southern Bahia-Brazil.

Paracoccidioidomycosis (PCM) cannot always be diagnosed by conventional means such as direct examination of histopathology or clinical samples, and serological methods, used as an alternative, still have many cases of cross-reactivity. In this scenario, molecular techniques seem to arise as a rapid approach, specific and direct that could be used in the diagnosis of this mycosis. In this study we analyzed 76 serum samples from patients in southern Bahia suspected of having paracoccidioidomycosis using a conventional PCR with primers for the ITS1 ribosomal DNA of P. brasiliensis. Of these 76 patients, 5 were positive for PCM by double immunodiffusion and/or direct examination and histopathology. To test specificity of PCR, we used human DNA and three isolates of P. lutzii (1578, 01 and ED01). Additionally, we analyzed by serial dilutions of DNA the limit of detection of the assay. The test of PCR proved specific, as only a 144 bp fragment of the three isolates of P. lutzii and no human DNA was amplified. Detection limit was 1.1 pg/µL of DNA. Despite the high detection limit and specificity of PCR none of the 76 serum samples were found positive by PCR, but a biopsy specimen obtained from one of the patients with PCM was positive. These results, albeit limited, show that PCR is not effective in detecting DNA of P. brasiliensis or P. lutzii in serum, but could perhaps be used with other types of clinical samples, especially in those instances in which conventional methods fail.

Comparison of a PCR assay in whole blood and serum specimens for canine brucellosis diagnosis.

The performance of a serum PCR assay was compared with that of a blood PCR assay for the diagnosis of canine brucellosis caused by Brucella canis in 72 dogs. The dogs were classified into three groups (infected, non-infected and suspected brucellosis) according to the results of blood culture and serological tests. The sensitivities of blood PCR and serum PCR were, respectively, 97.14 per cent and 25.71 per cent. The specificities of both were 100 per cent. In the group of dogs with suspected brucellosis, three were positive by blood PCR and none was positive by serum PCR. Serum PCR showed little value for the direct diagnosis of canine brucellosis as the assay had low diagnostic sensitivity and fewer positive dogs were detected by this test than by blood culture, blood PCR, rapid slide agglutination test (RSAT) and RSAT with 2-mercaptoethanol.

Susceptibility of Arcobacter butzleri to human blood serum / Susceptibilidade de Arcobacter butzleri ao soro humano

A susceptibilidade de 10 amostras de Arcobacter butzleri ao soro humano foi estudada. A maior atividade bactericida foi encontrada no soro humano normal, com taxas de sobrevivência bacterianainversamente proporcionais à diluição do soro. As maiores taxas de sobrevivência foram obtidas com o soro inativado pelo calor. As taxas de sobrevivência decresceram com a adição de soro fresco ao inativado. O soro com valores reduzidos de gamaglobulinas e valores normais de complemento mostrou ativo efeito bactericida. Os resultados demonstraram que A. butzleri é altamente susceptível ao efeito bactericida do soro humano, sugerindo que pode ser capaz de ativar diretamente o complemento pela via alternativa.(AU)

Susceptibility of Arcobacter butzleri to human blood serum / Susceptibilidade de Arcobacter butzleri ao soro humano

A susceptibilidade de 10 amostras de Arcobacter butzleri ao soro humano foi estudada. A maior atividade bactericida foi encontrada no soro humano normal, com taxas de sobrevivência bacterianainversamente proporcionais à diluição do soro. As maiores taxas de sobrevivência foram obtidas com o soro inativado pelo calor. As taxas de sobrevivência decresceram com a adição de soro fresco ao inativado. O soro com valores reduzidos de gamaglobulinas e valores normais de complemento mostrou ativo efeito bactericida. Os resultados demonstraram que A. butzleri é altamente susceptível ao efeito bactericida do soro humano, sugerindo que pode ser capaz de ativar diretamente o complemento pela via alternativa.

Isolation and characterization of Bartonella bacilliformis from an expatriate Ecuadorian.

Carrion's disease is typically biphasic with acute febrile illness characterized by bacteremia and severe hemolytic anemia (Oroya fever), followed by benign, chronic cutaneous lesions (verruga peruana). The causative agent, Bartonella bacilliformis, is endemic in specific regions of Peru and Ecuador. We describe atypical infection in an expatriate patient who presented with acute splenomegaly and anemia 3 years after visiting Ecuador. Initial serology and PCR of the patient's blood and serum were negative for Bartonella henselae, Bartonella quintana, and B. bacilliformis. Histology of splenic biopsy was suggestive of bacillary angiomatosis, but immunohistochemistry ruled out B. henselae and B. quintana. Bacilli (isolate EC-01) were subsequently cultured from the patient's blood and analyzed using multilocus sequence typing, protein gel electrophoresis with Western blotting, and an immunofluorescence assay (IFA) against a panel of sera from patients with Oroya fever in Peru. The EC-01 nucleotide sequences (gltA and internal transcribed spacer) and protein band banding pattern were most similar to a subset of B. bacilliformis isolates from the region of Caraz, Ancash, in Peru, where B. bacilliformis is endemic. By IFA, the patient's serum reacted strongly to two out of the three Peruvian B. bacilliformis isolates tested, and EC-01 antigen reacted with 13/20 Oroya fever sera. Bacilliary angiomatosis-like lesions were also detected in the spleen of the patient, who was inapparently infected with B. bacilliformis and who presumably acquired infection in a region of Ecuador where B. bacilliformis was not thought to be endemic. This study suggests that the range of B. bacilliformis may be expanding from areas of endemicity in Ecuador and that infection may present as atypical clinical disease.

Potential application of low-stringency single specific primer-PCR in the identification of Leptospira in the serum of patients with suspected leptospirosis.

In this study we tested the potential use of low-stringency single specific primer-PCR (LSSP-PCR) for genetically typing Leptospira directly from biological samples. Serum samples obtained from 29 patients with clinically suspected leptospirosis were amplified by specific PCR, using the previously selected G1 and G2 primers. The PCR products of approximately 300 bp were subsequently used as a template for LSSP-PCR analysis. We were able to produce genetic signatures from the leptospires present in the human samples, which permitted us to make a preliminary identification of the infective serovar by comparing the LSSP-PCR profiles obtained directly from serum samples with those from reference leptospires. Thus, LSSP-PCR has the potential to become a useful diagnostic tool for identifying leptospires in biological samples without the need for bacteria isolation and culture.