Differential expression of immune response genes associated with subclinical mastitis in dairy buffaloes.

Buffalo milk production has become of significant importance on the world scale, however, there are few studies involving biotechnological tools specifically for buffalo. To verify the effects caused by subclinical mastitis on the components of milk and to study the innate immune system in the udder of dairy buffaloes with subclinical mastitis, we evaluated the levels of expression of the lactoferrin (LTF), tumor necrosis factor alpha (TNF-α), interleukin-1 beta (IL-1ß), interleukin-8 (IL-8), and toll-like receptors 2 (TLR-2) and 4 (TLR-4) genes in buffaloes with and without subclinical mastitis. Milk samples were collected for the determination of milk components: somatic cell score (SCS), fat, protein, lactose, total solids and solids-not-fat (SNF), as well as for RNA extraction of milk cells, complementary DNA synthesis, and expression profile quantification by quantitative real-time PCR. For gene expression, the ΔΔCt was estimated using contrasts of the target genes expression adjusted for the expression of the housekeeping genes between both groups. Linear regression analysis was performed to determine the relationship between the genes studied and the milk components. Subclinical mastitis induced changes in the fat, lactose and SNF in milk of buffaloes, and the messenger RNA abundance was upregulated for TLR-2, TLR-4, TNF-α, IL-1ß and IL-8 genes in milk cells of buffaloes with subclinical mastitis, whereas the LTF gene was not differentially expressed. Results of linear regression analysis showed that TLR-2 gene expression most explains the variation in SCS, and the change in a unit of ΔCt of the TNF-α gene would result in a higher increase in SCS. The study of these immune function genes that are active in the mammary gland is important to characterize the action mechanism of the innate immunity that occurs in subclinical mastitis in dairy buffaloes and may aid the development of strategies to preserve the health of the udder.

Differential gene expression in drought-tolerant sugarcane roots.

Drought is one of the most frequent abiotic stresses limiting the productivity and geographical distribution of sugarcane culture. The use of drought-tolerant genotypes is one approach for overcoming the effects of water stress. We conducted a comparative study to identify gene expression profiles under water stress in tolerant sugarcane roots. Two different cultivars, 1 drought tolerant (RB867515) and 1 drought susceptible (SP86-155), were evaluated at 4 sampling time points (1, 3, 5, and 10 days) using the cDNA-amplified fragment length polymorphism technique. A total of 173 fragments were found to be differentially expressed in response to water stress in the tolerant cultivar. Seventy of these were cloned, sequenced, and categorized. Similarity analysis using BLAST revealed that 64% of the fragments differentially expressed code proteins classified as no hits (23%), hypothetical (21%), or involved in stress response (20%), with others were involved in communication pathways and signal transduction, bioenergetics, secondary metabolism, and growth and development. Four genes were analyzed and validated using real-time quantitative polymerase chain reaction to determine their expression and showed consistency with the cDNA-amplified fragment length polymorphism analyses. Our results contribute insight into the molecular responses to water stress in sugarcane and possibility to the development of cultivars with improved tolerance to drought.

The typing of botulinal neurotoxins.

The serological identification of Clostridium botulinum neurotoxin (BoNT) subtypes has shown to be elusive when current standard serologic tests are used. Based on (1) the in vivo response expected on quantitative BoNT-antitoxin systems and (2) the actual and the hypothetical antigenic makeup of BoNT subtypes, a comprehensive method for BoNTs typing is proposed.

Clostridium botulinum tipos D y A en materiales de necropsia de bovinos afectados por el mal de Aguapey / Clostridium botulinum type D and A in necropsy materials of bovines affected by Aguapey disease

Se realizaron exámenes bacteriológicos en materiales de necropsia de cinco animales muertos con diagnóstico clínico de botulismo bovino, enfermedad enzoótica regional llamada Mal de Aguapey (Provincia de Corrientes). Se identificó C. botulinum tipo D o su toxina en todos los animales, alternativamente en contenidos de rumen, yeyuno, íleon y ciego, y en muestras obtenidas de bazo, riñon e hígado. C. botulinum tipo A fue identificado, respectivamente, en hígado y riñón de dos animales. En los cultivos de cien muestras de diferentes tipos de suelo tomados en el área enzoótica, se identificó toxina butulínica sólo del tipo A en tres y en ninguna tipo D. Estos resultados amplían y confirman los hallazgos previos realizados sobre la etiología botulínica del Mal de Aguapey

Clostridium botulinum tipos D y A en materiales de necropsia de bovinos afectados por el mal de Aguapey / Clostridium botulinum type D and A in necropsy materials of bovines affected by Aguapey disease

Se realizaron exámenes bacteriológicos en materiales de necropsia de cinco animales muertos con diagnóstico clínico de botulismo bovino, enfermedad enzoótica regional llamada Mal de Aguapey (Provincia de Corrientes). Se identificó C. botulinum tipo D o su toxina en todos los animales, alternativamente en contenidos de rumen, yeyuno, íleon y ciego, y en muestras obtenidas de bazo, riñon e hígado. C. botulinum tipo A fue identificado, respectivamente, en hígado y riñón de dos animales. En los cultivos de cien muestras de diferentes tipos de suelo tomados en el área enzoótica, se identificó toxina butulínica sólo del tipo A en tres y en ninguna tipo D. Estos resultados amplían y confirman los hallazgos previos realizados sobre la etiología botulínica del Mal de Aguapey (AU)

[Type D and A Clostridium botulinum in necropsy samples from bovines with "mal de Aguapey"]. / Clostridium botulinum tipos D y A en materiales de necropsia de bovinos afectados por el mal de Aguapey.

Bacteriological studies were carried out on several necropsy samples from five animals whose deaths had been attributed to bovine botulism. This disease, regionally called Mal de Aguapey, enzootically affects animals from a wide area of the north-east of Argentina (Province of Corrientes) with a bovine population estimated at near to 2,500,000. Either C. botulinum type D, its toxin or both were identified in all animal samples, alternatively in contents of rumen, jejunum, ileum, caecum and in samples of spleen, liver and kidney (Table 1). C. botulinum type A was isolated respectively from the liver and the kidney of two animals. Cultures of 100 soil samples taken in the enzootic area were positive only for C. botulinum type A (3%). These results enlarge and confirm previous findings and lend support to the botulinic etiology of the Mal de Aguapey.

Clostridium botulinum tipos D y A en materiales de necropsia de bovinos afectados por el mal de Aguapey. / [Type D and A Clostridium botulinum in necropsy samples from bovines with [quot ]mal de Aguapey[quot ]]

Bacteriological studies were carried out on several necropsy samples from five animals whose deaths had been attributed to bovine botulism. This disease, regionally called Mal de Aguapey, enzootically affects animals from a wide area of the north-east of Argentina (Province of Corrientes) with a bovine population estimated at near to 2,500,000. Either C. botulinum type D, its toxin or both were identified in all animal samples, alternatively in contents of rumen, jejunum, ileum, caecum and in samples of spleen, liver and kidney (Table 1). C. botulinum type A was isolated respectively from the liver and the kidney of two animals. Cultures of 100 soil samples taken in the enzootic area were positive only for C. botulinum type A (3

). These results enlarge and confirm previous findings and lend support to the botulinic etiology of the Mal de Aguapey.

Prevalence and characterization of Staphylococcus aureus in young goats.

Thirty-six Staphylococcus aureus isolates recovered from 35 of 204 young goats at slaughter were characterized. All isolates were susceptible to cephalothin, clindamycin, chloramphenicol, gentamicin, kanamycin, and amikacin. All but 2 were susceptible to erythromycin and tetracycline, and 19 and 20 were susceptible to penicillin and ampicillin, respectively. Thirteen isolates were classified as biotype A, 9 isolates were classified as biotype B, 8 isolates were classified as biotype C, and 6 isolates were classified as intermediate between B and C or were not biotypable. Six biotype A isolates were enterotoxigenic; 4 produced enterotoxin B, 1 produced enterotoxin C, and 1 produced enterotoxin D. Two biotype B strains produced enterotoxin B, and all 8 biotype C isolates produced enterotoxin C and the toxic shock syndrome toxin-1.

[Growth of Clostridium botulinum in media with garlic (Allium sativum)]. / Crecimiento de Clostridium botulinum en medios con ajo (Allium sativum).

The effect of garlic on the growth and toxin formation of Clostridium botulinum (GT) was studied in A) crude juice obtained from a pool of cloves by i) crushing, ii) pressing out and iii) filtration, and B) minced garlic (6 to 8 pieces per clove). For both, "white" and "red" garlic varieties were used. The juice (pH 5.7 to 6.0, for different batches) was activated 30 min at 37 degrees C and diluted (log 2) in PGY broth (g%: peptone (Difco) 1.0; glucose 0.5; yeast extract (Difco) 0.5; pH 7.3). A small drop from a 18 h at 37 degrees C chopped meat medium culture of a highly toxigenic autochthonous strain (110) of C. botulinum type A, was transferred to the juice dilutions, incubating anaerobically 15d at 37 degrees C. As a control of the inhibitory effect of the juice, four microorganisms were cultured 48 h at 37 degrees C in the juice dilutions (Table 1). Clove pieces were suspended to 50% (w/v) either in PGY broth or distilled water without pH adjustment. Aliquots were heated in water bath 15 min at 100 degrees C. After seeded with the A 110 strain, duplicate tubes and their controls were incubated 15 d at 37 degrees C in aerated and anaerobic conditions (Table 2). Titers of botulinum toxin were empirically estimated by the time to death of a pair of mice injected with 0.5 ml each, via IP, observed 72 h. Results are shown in tables 1 and 2. Garlic reduces (in undiluted juice, traces or 3 to 5 DL50/ml were recorded in separate experiments) but not inhibit GT.(ABSTRACT TRUNCATED AT 250 WORDS)

Crecimiento de Clostridium botulinum en medios con ajo (Allium sativum). / [Growth of Clostridium botulinum in media with garlic (Allium sativum)]

The effect of garlic on the growth and toxin formation of Clostridium botulinum (GT) was studied in A) crude juice obtained from a pool of cloves by i) crushing, ii) pressing out and iii) filtration, and B) minced garlic (6 to 8 pieces per clove). For both, [quot ]white[quot ] and [quot ]red[quot ] garlic varieties were used. The juice (pH 5.7 to 6.0, for different batches) was activated 30 min at 37 degrees C and diluted (log 2) in PGY broth (g

: peptone (Difco) 1.0; glucose 0.5; yeast extract (Difco) 0.5; pH 7.3). A small drop from a 18 h at 37 degrees C chopped meat medium culture of a highly toxigenic autochthonous strain (110) of C. botulinum type A, was transferred to the juice dilutions, incubating anaerobically 15d at 37 degrees C. As a control of the inhibitory effect of the juice, four microorganisms were cultured 48 h at 37 degrees C in the juice dilutions (Table 1). Clove pieces were suspended to 50

(w/v) either in PGY broth or distilled water without pH adjustment. Aliquots were heated in water bath 15 min at 100 degrees C. After seeded with the A 110 strain, duplicate tubes and their controls were incubated 15 d at 37 degrees C in aerated and anaerobic conditions (Table 2). Titers of botulinum toxin were empirically estimated by the time to death of a pair of mice injected with 0.5 ml each, via IP, observed 72 h. Results are shown in tables 1 and 2. Garlic reduces (in undiluted juice, traces or 3 to 5 DL50/ml were recorded in separate experiments) but not inhibit GT.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of proteins, insoluble at low temperature, produced by Clostridium botulinum type C and C. subterminale. Their antigenic relationship with a similar protein synthesized by C. botulinum type G.

The production of a protein insoluble at low temperature ("cryoprotein"), by cultures of Clostridium botulinum type G has been shown to be a metabolic characteristic also shared by C. botulinum type C and by C. subterminale. These new cryoproteins have been purified and some of their chemical and immunological properties studied. It was found that both proteins were chemically very similar among themselves and to the cryoprotein isolated from C. botulinum type G. All these proteins are formed by a single polypeptide chain of approximately Mr = 180,000, with closely related amino acid compositions, isoelectric points and do not contain either free cysteine or disulfide bridges. Homologous and heterologous radioimmunoassays established the existence of an antigenic similitude among the cryoproteins from C. botulinum type G and C. subterminale thus becoming the first purified antigens which relate both bacterial species. If the production of cryoproteins can be shown to be a generalized phenomenon within the genus Clostridium these substances would provide an important tool to examine immunological and genetical relatedness between strains in this bacterial group.

[[Staphylococcus aureus producing toxic shock syndrome toxin 1 in the lower genital tract of the female]. / Staphylococcus aureus productores de toxina del síndrome del shock tóxico-1 en el tracto genital inferior femenino.

Staphylococcus sp was investigated in the female lower genital tract of 102 healthy women aged between 18 and 48 years in San Luis, Argentina. Three hundred and six samples were obtained from labia, introitus and vagina (posterior fornix). Samples were plated on sheep blood, mannitol salt and Baird-Parker media. Strains were identified by tube coagulase test; thermonuclease, fibrinolysin, pigment and hemolysin production; glucose and mannitol utilization and novobiocin sensitivity. Antibiotic susceptibility was assayed. Strains were examined for their ability to produce staphylococcal enterotoxins (SE) and toxic shock syndrome toxin-1 (TSST-1). Fourteen women (13.7%) had S. aureus in one or more samples: 10.7% labia, 3.9% introitus and 3.9% vaginal. All strains were sensitive to cephalotin, clindamycin, erythromycin, gentamycin and chloramphenicol; 21.0% were intermediate to methicillin; 15.7% were resistant to methicillin, 94.7% to penicillin and 21.0% to tetracycline. Three strains (15.7%) produced SEB, three (15.7%) SED, one (5.7%) SEC and three (15.7%) TSST-1. Only one strain (5.7%) produced both SEB and TSST-1. All strains produced hemolysins. Coagulase negative staphylococci were found in 40.1% of vaginal samples: S. epidermidis (32.2%) and S. saprophyticus (9.8%) were identified.

Rapid in vivo assay method for type G botulinal toxin.

The survival time (ST) dose-response curve for Clostridium botulinum type G toxin was determined in mice and evaluated as a rapid assay method. As it occurs with other botulinal toxin types, the results showed a linear relationship between the logarithm of the injected dose and the logarithm of ST. The slope of the ST dose-response curve for type G toxin differed significantly from those for type A or subtype Af toxins. This parameter was altered when trypsin-activated type G toxin was used. The ST dose-response curve was rather stable. This in vivo assay method could be applied for the estimation of the potency of G toxin preparations in a short time using only some few mice.

Staphylococcus aureus productores de toxina del síndrome del shock tóxico-1 en el tracto genital inferior femenino. / [[Staphylococcus aureus producing toxic shock syndrome toxin 1 in the lower genital tract of the female]

Staphylococcus sp was investigated in the female lower genital tract of 102 healthy women aged between 18 and 48 years in San Luis, Argentina. Three hundred and six samples were obtained from labia, introitus and vagina (posterior fornix). Samples were plated on sheep blood, mannitol salt and Baird-Parker media. Strains were identified by tube coagulase test; thermonuclease, fibrinolysin, pigment and hemolysin production; glucose and mannitol utilization and novobiocin sensitivity. Antibiotic susceptibility was assayed. Strains were examined for their ability to produce staphylococcal enterotoxins (SE) and toxic shock syndrome toxin-1 (TSST-1). Fourteen women (13.7

) had S. aureus in one or more samples: 10.7

labia, 3.9

introitus and 3.9

vaginal. All strains were sensitive to cephalotin, clindamycin, erythromycin, gentamycin and chloramphenicol; 21.0

were intermediate to methicillin; 15.7

were resistant to methicillin, 94.7

to penicillin and 21.0

to tetracycline. Three strains (15.7

) produced SEB, three (15.7

) SED, one (5.7

) SEC and three (15.7

) TSST-1. Only one strain (5.7

) produced both SEB and TSST-1. All strains produced hemolysins. Coagulase negative staphylococci were found in 40.1

of vaginal samples: S. epidermidis (32.2

) and S. saprophyticus (9.8

) were identified.

[First outbreak of botulism caused by Clostridium botulinum subtype Af]. / Primer brote de botulismo por Clostridium botulinum subtipo Af.

In December 1982 an outbreak of foodborne botulism presumably produced by the ingestion of home-made pickled trout occurred in San Rafael, province of Mendoza, Argentina. The toxin detected in blood serum and feces samples of the sole affected patient was preliminarily typed as a plain type A. A strain of Clostridium botulinum was isolated from feces which, after culture by the dialysis method, produced 1 x 10(7) LD50/mouse per ml. Quantitative neutralization tests carried out at different levels of toxin concentration, showed that this toxin consists of a major type A antigenic component (about 99% of the complex) and a minor type F component, defining its identity as a subtype Af. A rabbit antiserum obtained from this toxin neutralizes A and F reference toxins. Despite the antitoxic and supportive treatment, the patient died as a consequence of the poisoning. The polyvalent antiserum administered contained A, B and E antitoxins. Death could be due to the lethal effect of the A fraction, the F fraction or to a combined effect of both toxic components of the toxin. This is the first detection of an outbreak produced by C. botulinum subtype Af, serotype described in Argentina fifteen years ago, and not detected until now in other parts of the world.(ABSTRACT TRUNCATED AT 250 WORDS)

Primer brote de botulismo por Clostridium botulinum subtipo Af. / [First outbreak of botulism caused by Clostridium botulinum subtype Af]

In December 1982 an outbreak of foodborne botulism presumably produced by the ingestion of home-made pickled trout occurred in San Rafael, province of Mendoza, Argentina. The toxin detected in blood serum and feces samples of the sole affected patient was preliminarily typed as a plain type A. A strain of Clostridium botulinum was isolated from feces which, after culture by the dialysis method, produced 1 x 10(7) LD50/mouse per ml. Quantitative neutralization tests carried out at different levels of toxin concentration, showed that this toxin consists of a major type A antigenic component (about 99

of the complex) and a minor type F component, defining its identity as a subtype Af. A rabbit antiserum obtained from this toxin neutralizes A and F reference toxins. Despite the antitoxic and supportive treatment, the patient died as a consequence of the poisoning. The polyvalent antiserum administered contained A, B and E antitoxins. Death could be due to the lethal effect of the A fraction, the F fraction or to a combined effect of both toxic components of the toxin. This is the first detection of an outbreak produced by C. botulinum subtype Af, serotype described in Argentina fifteen years ago, and not detected until now in other parts of the world.(ABSTRACT TRUNCATED AT 250 WORDS)

[Virulence and enterotoxigenicity of Yersinia enterocolitica isolated from foods and other biological material]. / Virulencia y enterotoxigenicidad de Yersinia enterocolitica aislada de alimentos y otros materiales biológicos.

Enterotoxigenicity, virulence and antimicrobial susceptibility were studied in thirteen strains of Yersinia enterocolitica isolated from meat foods and organs of slaughtered animals. The following biotypes (B), serotypes (O) and lisotypes (Lis) were studied: B2, O:9, Lis X3 and B1, O:7,8, Lis Xo (from thick fresh sausages); B1, O:5, Lis Xz (from slender sausages); B1, O:5, Lis Xz and B2, O:9, Lis X3 (from bovine tongues); B1, O:6, Lis Xz (from porcine tongues) and B1, O:6, Lis Xz (from porcine caeca) (Table 1). Virulence was determined by i) the autoagglutination test (Laird and Cavanaugh), replacing in the Eagle medium amino acids and vitamins with proteose peptone (1%), tryptone (1%) and yeast extract (0.5%), and ii) the calcium dependency test. The LD50 of five strains was determined by i.p. injection of O.5 ml of cell suspensions into white mice and estimated by the Spearman Kärber method. LD50 varied from 2 x 10(9) to 7 x 10(9) viable cells per ml. The enterotoxigenicity test was carried out in suckling white mice and the invasiveness test (Sereny) in adult white mice. Two strains showed autoagglutination, five calcium dependency, two were invasive and eight produced enterotoxin. Antimicrobial susceptibility was assayed by the modified Kirby and Bauer test. All the strains were susceptible to kanamycin, amikacin, cefotaxime, cefuroxime, chloramphenicol, fosfomycin, mezlocillin, nitrofurantoin, piperacillin, piperimidic acid, polymyxin B, sisomycin and tobramycin, and resistant to cephalotoxin, ampicillin and trimethoprim-sulfamethoxazole.(ABSTRACT TRUNCATED AT 250 WORDS)

Virulencia y enterotoxigenicidad de Yersinia enterocolitica aislada de alimentos y otros materiales biológicos / [Virulence and enterotoxigenicity of Yersinia enterocolitica isolated from foods and other biological material]

Enterotoxigenicity, virulence and antimicrobial susceptibility were studied in thirteen strains of Yersinia enterocolitica isolated from meat foods and organs of slaughtered animals. The following biotypes (B), serotypes (O) and lisotypes (Lis) were studied: B2, O:9, Lis X3 and B1, O:7,8, Lis Xo (from thick fresh sausages); B1, O:5, Lis Xz (from slender sausages); B1, O:5, Lis Xz and B2, O:9, Lis X3 (from bovine tongues); B1, O:6, Lis Xz (from porcine tongues) and B1, O:6, Lis Xz (from porcine caeca) (Table 1). Virulence was determined by i) the autoagglutination test (Laird and Cavanaugh), replacing in the Eagle medium amino acids and vitamins with proteose peptone (1

) and yeast extract (0.5

), and ii) the calcium dependency test. The LD50 of five strains was determined by i.p. injection of O.5 ml of cell suspensions into white mice and estimated by the Spearman Kõrber method. LD50 varied from 2 x 10(9) to 7 x 10(9) viable cells per ml. The enterotoxigenicity test was carried out in suckling white mice and the invasiveness test (Sereny) in adult white mice. Two strains showed autoagglutination, five calcium dependency, two were invasive and eight produced enterotoxin. Antimicrobial susceptibility was assayed by the modified Kirby and Bauer test. All the strains were susceptible to kanamycin, amikacin, cefotaxime, cefuroxime, chloramphenicol, fosfomycin, mezlocillin, nitrofurantoin, piperacillin, piperimidic acid, polymyxin B, sisomycin and tobramycin, and resistant to cephalotoxin, ampicillin and trimethoprim-sulfamethoxazole.(ABSTRACT TRUNCATED AT 250 WORDS)

Virulencia y enterotoxigenicidad de Yersinia enterocolitica aislada de alimentos y otros materiales biológicos. / [Virulence and enterotoxigenicity of Yersinia enterocolitica isolated from foods and other biological material]

Enterotoxigenicity, virulence and antimicrobial susceptibility were studied in thirteen strains of Yersinia enterocolitica isolated from meat foods and organs of slaughtered animals. The following biotypes (B), serotypes (O) and lisotypes (Lis) were studied: B2, O:9, Lis X3 and B1, O:7,8, Lis Xo (from thick fresh sausages); B1, O:5, Lis Xz (from slender sausages); B1, O:5, Lis Xz and B2, O:9, Lis X3 (from bovine tongues); B1, O:6, Lis Xz (from porcine tongues) and B1, O:6, Lis Xz (from porcine caeca) (Table 1). Virulence was determined by i) the autoagglutination test (Laird and Cavanaugh), replacing in the Eagle medium amino acids and vitamins with proteose peptone (1

), tryptone (1

) and yeast extract (0.5

), and ii) the calcium dependency test. The LD50 of five strains was determined by i.p. injection of O.5 ml of cell suspensions into white mice and estimated by the Spearman Kõrber method. LD50 varied from 2 x 10(9) to 7 x 10(9) viable cells per ml. The enterotoxigenicity test was carried out in suckling white mice and the invasiveness test (Sereny) in adult white mice. Two strains showed autoagglutination, five calcium dependency, two were invasive and eight produced enterotoxin. Antimicrobial susceptibility was assayed by the modified Kirby and Bauer test. All the strains were susceptible to kanamycin, amikacin, cefotaxime, cefuroxime, chloramphenicol, fosfomycin, mezlocillin, nitrofurantoin, piperacillin, piperimidic acid, polymyxin B, sisomycin and tobramycin, and resistant to cephalotoxin, ampicillin and trimethoprim-sulfamethoxazole.(ABSTRACT TRUNCATED AT 250 WORDS)