Fowl typhoid: assessment of a disinfectant oral dose to reduce horizontal spread and mortality.

To determine the most appropriate dose in drinking water of the disinfectant N-alkyl dimethyl benzyl ammonium chloride (TIMSEN), a fowl typhoid challenge trial was carried out using 21-day-old Salmonella-free chickens. In a pretrial, performed with six groups of 10 chickens each, it was shown that the disinfectant was atoxic and safe when administered during 15 days at doses of 0 ppm, 200 ppm, 400 ppm, 800 ppm, 1600 ppm, or 3200 ppm. Thereafter, a challenge trial was performed with 390 chickens divided into six groups of 65 birds each. Chickens were treated during 9 days with oral doses of 0 ppm, 25 ppm, 50 ppm, 100 ppm, 250 ppm, and 500 ppm (groups identified as A, B, C, D, E, and F, respectively). Twenty-four hours after the beginning of the treatment, 30 chickens of each group were orally inoculated with 10(9) colony-forming units of Salmonella Gallinarum strain INTA 91 per bird. The remaining 35 unchallenged chickens from each group were left in the same cage in close contact with the other challenged birds of their group. All surviving chickens were sacrificed 8 days after challenge. Livers from all birds were examined for the presence of Salmonella Gallinarum. Salmonella Gallinarum was isolated from all challenged chickens and from some unchallenged chickens of groups A (4/35), E (3/35), and F (6/35), but not from any unchallenged chicken from groups B, C, and D. Doses of 50 ppm (group C) significantly reduced mortality (30%) in comparison with the untreated control group A (65%). Mortality in group B (45%) was not significantly different from group C. Administration of higher doses of the disinfectant resulted in significantly higher mortality rates, 70% in group E and 85% in groups D and F. Increased infection and mortality rates of groups D, E, and F might have been caused by inhibition of the protective action of the normal gut flora. To reduce horizontal infection and mortality, the manufacturer's prescribed oral dose of 25 ppm may be increased up to 50 ppm. Nevertheless, higher doses should be avoided because they may cause horizontal increased spread of the disease and mortality.

Protection conferred by a live Salmonella Enteritidis vaccine against fowl typhoid in laying hens.

Fowl typhoid is under control in poultry farms of developed countries, but it still endemically subsists in commercial laying hen farms of some countries. It has been demonstrated that Salmonella live vaccines can elicit cross-immunity against members of the same Kauffmann-White scheme serogroup. In this work, we explored the protection conferred by TAD Salmonella vac E, a live Salmonella enterica serovar Enteritidis vaccine, against fowl typhoid. Three groups of laying hens were vaccinated with different vaccination schedules starting on the first day of life, and afterwards were infected with 2 x 10(5) CFU of a virulent Salmonella Gallinarum strain, either at wk 28 or wk 52. Mortality, fecal shedding, and organ invasion of Salmonella Gallinarum were assessed. In this work we demonstrated that this Salmonella Enteritidis vaccine is able to cross-immunize against Salmonella Gallinarum. At wk 28, hens vaccinated with three oral doses or with two oral doses combined with one subcutaneous dose were protected by the vaccine. At wk 52, when hens were infected 36 wk after the final immunization, the vaccine was not able to confer protection. Thus, revaccination every 3 mo would be highly recommended. In countries where Salmonella Gallinarum subsists together with Salmonella Enteritidis, control programs should include vaccination of laying hens using safe attenuated Salmonella strains.

[Agglutination of hen egg-yolk immunoglobulins (IgY) against Salmonella enterica, serovar enteritidis]. / Aglutinación de inmunoglobulinas de yema de huevo de gallina (IgY) contra Salmonella enterica serovariedad enteritidis.

Two groups of 6 laying hens were used to produce IgY. In the vaccinated group (V), hens were injected by intramuscular route with two doses of a Salmonella enterica serovar Enteritidis bacterin at 20-day interval. In the control group (T) hens remained unvaccinated. Four IgY extractions were performed on the egg production of both groups. The first two extractions were carried out using the yolks obtained from the eggs produced during the 4th and 5th post-vaccination week (extracts 1V and 1T) and the other two using the ones from the 6th, 7th and 8th week (2V and 2T). Starting from the extracts 1V and 1T other products were obtained by freezing-thawing (1V-A and 1T-A) and simple (1V-B and 1T-B) or double (1V-C and 1T-C) flow capillary dialysis concentration. All these products were compared using an ELISA test specific for the detection of chicken antibodies against flagellar antigens of S. Enteritidis. In this test, V extracts were positive whereas T extracts were negative. The extract 1V was more positive than the extract 2V. The extract 1V-C was the most positive and was therefore selected to be used as an antiserum in the agglutination tests. This extract contained 1.9 g/dl of total proteins, 0.028 g/dl of triglycerides and 0.012 g/dl of cholesterol and showed an electrophoretic pattern characteristic of IgY. The 1T-C extract was used as a negative control in the agglutination tests. Slide somatic and tube flagellar agglutination tests were simultaneously carried out using both IgY extracts and a standard rabbit anti-Salmonella (IgG) sera. Overall 367 strains from the Enterobacteriaceae family were tested together with two other strains belonging to the Vibrionaceae family. The 1V-C extract specifically agglutinated S. Enteritidis strains in the same way as the rabbit sera. This extract also agglutinated other Salmonella strains antigenically related to S. Enteritidis. Salmonella which did not share somatic or flagellar antigens with S. Enteritidis, other different species of the Enterobacteriaceae family and the two strains of the Vibrionaceae family were all negative. None of the strains tested was agglutinated by the 1T-C extract. This paper show that it is possible to use specific IgY to identify S. enterica serovars. The more extended use of IgY for diagnostic purposes may be a convenient way to complement the current use of mammal polyclonal antibodies.

Aglutinación de inmunoglobulinas de yema de huevo de gallina (IgY) contra Salmonella enterica serovariedad Enteritidis / Agglutination of chicken egg yolk immunoglobulins (IgY) against Salmonella enterica serovar Enteriditis

Para producir extractos de IgY se emplearon dos lotes de 6 gallinas ponedoras cada uno. En el lote vacunado (V) las aves se inocularon por vía intramuscular con dos dosis de una bacterina contra Salmonella enterica serovariedad Enteritidis. En el lote testigo (T) las aves no se vacunaron. Con las yemas de ambos lotes se efectuaron 4 extracciones de IgY. Las dos primeras se realizaron con las yemas de los huevos producidos durante la 4§ y 5§ semana post-vacunación (extractos 1V y 1T) y las otras dos con las de la 6§, 7§ y 8§ semana (2V y 2T). Los extractos 1V y 1T se congelaton y descongelaron (1V-A y 1T-A) y se concentraron por diálisis simple (1V-B y 1T-B) o doble (1V-C y 1T-C). Mediante una prueba de ELISA para detectar antígenos flagelares de S. Enteritidis, los extractos V fueron positivos y los T negativos. El extracto 1V-C fue el más positivo y se seleccionó para realizar las aglutinaciones. Este extracto contenía 1,9 g/dl de proteínas totales y presentó bandas electroforéticas características de IgY. El extracto 1T-C fue usado como control negativo de aglutinación. Emplenado ambos extractos IgY y antisueros policlonales de conejo (IgG) se efectuaron aglutinaciones somáticas en placa y flagelares en tubo. Se estudiaron 357 cepas de S. enterica, 10 cepas de diferentes especies de la familia Enterobacteriaceae y dos cepas de la familia Vibrionaceae. El extracto 1V-C aglutinó a distintas cepas de Salmonella con estructura antigénica somática o flagelar relacionada con la cepa vacunal, mientras que las salmonelas con antígenos diferentes y otras especies de la familia Enterobacteriaceae y Vibrionaceae fueron negativas. Este trabajo demuestra que es posible emplear las IgY para identificar serovariedades de S. enterica

Aglutinación de inmunoglobulinas de yema de huevo de gallina (IgY) contra Salmonella enterica serovariedad Enteritidis / Agglutination of chicken egg yolk immunoglobulins (IgY) against Salmonella enterica serovar Enteriditis

Para producir extractos de IgY se emplearon dos lotes de 6 gallinas ponedoras cada uno. En el lote vacunado (V) las aves se inocularon por vía intramuscular con dos dosis de una bacterina contra Salmonella enterica serovariedad Enteritidis. En el lote testigo (T) las aves no se vacunaron. Con las yemas de ambos lotes se efectuaron 4 extracciones de IgY. Las dos primeras se realizaron con las yemas de los huevos producidos durante la 4º y 5º semana post-vacunación (extractos 1V y 1T) y las otras dos con las de la 6º, 7º y 8º semana (2V y 2T). Los extractos 1V y 1T se congelaton y descongelaron (1V-A y 1T-A) y se concentraron por diálisis simple (1V-B y 1T-B) o doble (1V-C y 1T-C). Media

Evaluation of inactivated infectious coryza vaccines in chickens challenged by serovar B strains of Haemophilus paragallinarum.

Four monovalent experimental vaccines (VI, V2, V3 and V7) containing an Argentinean serovar B strain (H8) of Haemophilus paragallinarum and three different commercial vaccines, either bivalent (V4 and V5) containing serovars A and C, or trivalent (V6) containing serovars A, B and C were administered by subcutaneous or intramuscular routes as a single or double dose (at 3-week intervals) to chickens of between 6 and 10 weeks. Three to 7 weeks after the last vaccination, vaccinated and non-vaccinated chickens were challenged by intrasinus inoculation with Argentinean serovar B strains of H. paragallinarum. When the vaccinated chickens were exposed to a severe challenge with the vaccinal strain (H8) some experimental vaccines protected, whereas all commercial vaccines failed to protect. The experimental vaccines manufactured in broth (V2, V3 and V7) protected more effectively than the vaccine produced in chicken embryos (VI). Failure of the commercial trivalent vaccine V6 to protect may be related to the method of manufacture. Vaccine V7 protected against challenge from either the vaccinal strain (H8) or three Argentinean serovar B strains (H6, Hll and HI2). These results confirm the necessity of including serovar B regional strains in the formulation of local vaccines.

Evaluación de medios de cultivo selectivos para el aislamiento de Salmonella en producción avícola / Evaluation of selective culture media for isolation of Salmonella from poultry

Se evaluó el desempeño de los medios agar Rambach, agar xilosa-lisina-desoxicolato (XLD) con el agregado de diferentes concentraciones de tergitol tipo 4 o sulfato de sodio y de 7-etil-2-metil-4-undecanol (XLDT4), agar Salmonella-Shigella (SS) y agar sulfito de bismuto según Wilson-Blair (SB) utilizando serovariedades de Salmonella spp. y otras especies bacterianas de la flora intestinal de las aves. Los medios de cultivo selectivos fueron evaluados mediante recuento de bacterias viables, comparando estos resultados con los del agar base Columbia (ABC) adicionado con sangre bovina al 7 por ciento, para lo cual se emplearon las serovariedades de Salmonella spp. que con mayor frecuencia se aíslan en las aves de nuestro país. Además se analizaron muestras provenientes de pollos experimentalmente inoculados con distintas serovariedades de Salmonella. Los medios Rambach, SS y XLD o XLDT4 son adecuados para el aislamiento de salmonelas. En cambio el agar SB fue muy inhibidor para las salmonelas de interés veterinario. El agregado de novobiocina o tergitol al medio XLD no inhibio completamente a todas las cepas de Proteus. En el agar Rambach comercial no creció ninguna de las cepas de Proteus que habían desarrollado en los otros medios. Diversas bacterias contaminantes produjeron colonias similares a Salmonella en los agares Rambach, SS, XLD y XLDT4. Dado que las especies bacterianas contaminantes que desarrollan en los medios de cultivo son diferentes, es recomendable optimizar el diagnóstico sembrando las muestras en los agares SS, XLD o XLDT4 y simultáneamente también en agar Rambach

Evaluación de medios de cultivo selectivos para el aislamiento de Salmonella en producción avícola / Evaluation of selective culture media for isolation of Salmonella from poultry

Se evaluó el desempeño de los medios agar Rambach, agar xilosa-lisina-desoxicolato (XLD) con el agregado de diferentes concentraciones de tergitol tipo 4 o sulfato de sodio y de 7-etil-2-metil-4-undecanol (XLDT4), agar Salmonella-Shigella (SS) y agar sulfito de bismuto según Wilson-Blair (SB) utilizando serovariedades de Salmonella spp. y otras especies bacterianas de la flora intestinal de las aves. Los medios de cultivo selectivos fueron evaluados mediante recuento de bacterias viables, comparando estos resultados con los del agar base Columbia (ABC) adicionado con sangre bovina al 7 por ciento, para lo cual se emplearon las serovariedades de Salmonella spp. que con mayor frecuencia se aíslan en las aves de nuestro país. Además se analizaron muestras provenientes de pollos experimentalmente inoculados con distintas serovariedades de Salmonella. Los medios Rambach, SS y XLD o XLDT4 son adecuados para el aislamiento de salmonelas. En cambio el agar SB fue muy inhibidor para las salmonelas de interés veterinario. El agregado de novobiocina o tergitol al medio XLD no inhibio completamente a todas las cepas de Proteus. En el agar Rambach comercial no creció ninguna de las cepas de Proteus que habían desarrollado en los otros medios. Diversas bacterias contaminantes produjeron colonias similares a Salmonella en los agares Rambach, SS, XLD y XLDT4. Dado que las especies bacterianas contaminantes que desarrollan en los medios de cultivo son diferentes, es recomendable optimizar el diagnóstico sembrando las muestras en los agares SS, XLD o XLDT4 y simultáneamente también en agar Rambach (AU)

[Evaluation of selective culture media for the isolation of Salmonella from poultry]. / Evaluación de medios de cultivo selectivos para el aislamiento de Salmonella en producción avícola.

Rambach agar, xylose-lysine-deoxycholate agar (XLD) with different concentrations of Tergitol 4 or 7 ethyl-2 methyl-4 undecanol hydrogen sulphate, sodium salt (XLDT4), Salmonella-Shigella agar (SS) and bismuth sulfite agar according to Wilson-Blair (BS) were evaluated using Salmonella spp. serovars and other bacterial species from the intestinal flora of poultry. Growth of the most common Salmonella serovars isolated from chickens in our country were evaluated using a viable counting technique on the different selective media and these results were compared with those obtained on Columbia base (ABC) agar plus 7% bovine blood (Table 1). Samples from Salmonella experimentally inoculated chickens were also examined. Results showed that Rambach, SS and XLD or XLDT4 were all satisfactory for isolation of Salmonella. Bismuth Sulfite agar was too inhibitory for bacteria important in veterinary practice. The characteristic colonies of Salmonella and other common fecal contaminant bacteria growing on SS, Rambach, XLDT4 and SB are shown in Table 2. Addition of tergitol or novobiocin to XLD agar did not completely inhibit the growth of all Proteus spp. strains examined. None of the Proteus spp. strains able to multiply on SS, XLD or XLDT4 agar grew on the commercial Rambach agar. Several different contaminant bacterial species produced Salmonella-like colonies on Rambach, SS, XLD and XLDT4 agars. Because these contaminant bacterial species are different it is advisable to improve the diagnosis by culturing samples on SS, XLD or XLDT4 agar and also simultaneously on Rambach agar.

Evaluación de medios de cultivo selectivos para el aislamiento de Salmonella en producción avícola. / [Evaluation of selective culture media for the isolation of Salmonella from poultry]

Rambach agar, xylose-lysine-deoxycholate agar (XLD) with different concentrations of Tergitol 4 or 7 ethyl-2 methyl-4 undecanol hydrogen sulphate, sodium salt (XLDT4), Salmonella-Shigella agar (SS) and bismuth sulfite agar according to Wilson-Blair (BS) were evaluated using Salmonella spp. serovars and other bacterial species from the intestinal flora of poultry. Growth of the most common Salmonella serovars isolated from chickens in our country were evaluated using a viable counting technique on the different selective media and these results were compared with those obtained on Columbia base (ABC) agar plus 7

bovine blood (Table 1). Samples from Salmonella experimentally inoculated chickens were also examined. Results showed that Rambach, SS and XLD or XLDT4 were all satisfactory for isolation of Salmonella. Bismuth Sulfite agar was too inhibitory for bacteria important in veterinary practice. The characteristic colonies of Salmonella and other common fecal contaminant bacteria growing on SS, Rambach, XLDT4 and SB are shown in Table 2. Addition of tergitol or novobiocin to XLD agar did not completely inhibit the growth of all Proteus spp. strains examined. None of the Proteus spp. strains able to multiply on SS, XLD or XLDT4 agar grew on the commercial Rambach agar. Several different contaminant bacterial species produced Salmonella-like colonies on Rambach, SS, XLD and XLDT4 agars. Because these contaminant bacterial species are different it is advisable to improve the diagnosis by culturing samples on SS, XLD or XLDT4 agar and also simultaneously on Rambach agar.

Complicated infectious coryza outbreaks in Argentina.

Seventeen complicated outbreaks of infectious coryza in layer, broiler-breeder, and broiler flocks were studied. In the layer flock outbreaks, drops in egg production of up to 35% were seen. In the broiler flocks and several of the layer flocks, losses due to persistent mortality and/or culling varied between 2 and 5%. Signs of infectious coryza in both layers and broiler-breeders were typical; in broilers, however, swollen head-like syndrome was seen. Except in one flock, no viral diseases were clinically or serologically detected. Excluding broiler-breeders, birds from most other flocks were serologically positive for Mycoplasma gallisepticum, and some were also positive for M. synoviae. Haemophilus paragallinarum was isolated from all of the outbreaks, but only as a pure culture in three outbreaks. Isolation of H. paragallinarum from sites such as liver, kidney, and particularly tarsal arthritis and ocular globes appears to be reported for the first time. Serovar A was isolated in eight outbreaks, serovar B in six, serovar C in one, and untypable serovars in two. The severity of these infectious coryza outbreaks may have been increased by concurrent salmonellosis, pasteurellosis, and mycoplasmosis, although under certain conditions H. paragallinarum is able to cause septicemia. Ten of the outbreaks occurred in birds vaccinated against infectious coryza; this may be due to the use of vaccines that do not provide protection against the types of H. paragallinarum that affect poultry in the region.

Characterization of two monoclonal antibodies directed against serovar A Haemophilus paragallinarum.

Two monoclonal antibodies (MAbs) raised against a serovar A Haemophilus paragallinarum were evaluated for their ability to react with 11 reference strains that represented all the recognized serovars and with 27 field isolates of Page serovar A collected from around the world. The MAbs were used in a hemagglutination-inhibition assay. Both MAbs recognized type strains of Page serovar A and Kume serovars A-1 and A-2 but not the type strains of Kume serovars A-3 and A-4. Neither MAb recognized the type strains of Page serovars B and C or Kume serovars B-1, C-1, C-2, C-3, or C-4. When evaluated with the 27 Page serovar A field isolates, both MAbs recognized only 10 isolates. All of the recognized isolates belonged to Kume serovars A-1 (nine isolates) or A-2 (one isolate). All of the field isolates that were not recognized by one or the other of the MAbs either were Kume serovar A-4 (seven isolates) or could not be placed in an existing Kume A serovar (10 isolates). The results indicate that the epitope recognized by these MAbs is present only in strains of H. paragallinarum that belong to Kume serovars A-1 and A-2.

Characterization of isolates of Haemophilus paragallinarum from Argentina.

The biochemical and serological properties of Haemophilus paragallinarum isolates recovered from 11 recent outbreaks of infectious coryza in layer hens and one case of swollen-head syndrome in broilers in Argentina are described. Twenty-four isolates had the typical biochemical properties of H. paragallinarum. All isolates were serotyped according to the Page scheme. Ten of the isolates were serovar A, 11 were serovar B, one was serovar C, and two isolates could not be serotyped. The isolates were also examined using a panel of monoclonal antibodies (MAbs) for Page serovars A (one MAb available) and C (three MAbs available). The serovar B isolates all failed to react with any MAb. The serovar C isolate reacted with all three serovar C MAbs but not with the serovar A MAb. Only six of the 10 serovar A isolates reacted with the serovar A MAb. These results indicate that H. paragallinarum isolates from Argentina are antigenically distinct from those examined in other countries, and it is suggested that coryza vaccines intended for use in Argentina may be more effective if based on local strains.

[Evaluation of antimicrobial drugs and atmospheres for the isolation of Campylobacter fetus subspp. from the bovine genital tract]. / Evaluación de drogas antimicrobianas y atmósferas para el aislamiento de Campylobacter fetus subspp. del tracto genital bovino.

Growth of 3 reference bovine C. fetus strains in media with and without antibiotics and bacteriostats active against the most common contaminant bacteria in the bovine genital tract was evaluated. In addition, 2 regional bovine C. fetus strains and 1 reference C. sputorum biovar bubulus strain were used in some experiments. Reference strain C. fetus subsp. venerealis was completely inhibited by polymyxin (> or = 0.25 IU/ml) whereas the other C. fetus strains were not inhibited. In Shepler's medium supplemented with rifampicin (10 micrograms/ml) subsp. fetus, was the only one to grow. When rifampicin was used at 5 micrograms/ml together with a reduced dose of the other Shepler's antibiotics, especially polymyxin B (0.85 IU/ml), subsp. venerealis was able to grow; nevertheless even at such a reduced dose, rifampicin was inhibitory for the biotype intermedius. It was demonstrated that triclosan (Irgasan) could be very useful at < or = 10 micrograms/ml in media with added blood, < or = 6 micrograms/ml in brucella broth and < or = 3 micrograms/ml in Mueller-Hinton broth for isolation of all subspecies of C. fetus. The sensitivity of C. fetus to 5-fluorouracil was variable: subsp. fetus was resistant (up to 800 micrograms/ml) whereas subspp. venerealis and biotype intermedius grew slowly or sometimes did not grow at all in concentrations of 6.25 micrograms/ml onwards. Fosfomycin was inhibitory to all C. fetus strains at > or = 50 micrograms/ml. C. sputorum biovar. bubulus was less inhibited than C. fetus with triclosan grew up to more than 100 micrograms/ml, with 5-fluorouracil up to 100 micrograms/ml and with fosfomycin up to 50 micrograms/ml. Growth of C. fetus subspp. was compared in different microaerophilic atmospheres contained in anaerobic jars (Oxoid HP 11) without palladium catalyzer. Growth with nitrogen or hydrogen was similar. When jars were replaced by 15 x 13 cm cylindrical cans without valves or gas measurement devices only pure hydrogen supported satisfactory growth of all C. fetus subspp. and C. sputorum biovar. bubulus strains. The candle system, a commercial nitrogen rich gas mixture and pure carbonic anhydride prepared in these cans failed to enable these strains to grow. C. fetus subsp. fetus was more aero-tolerant than subsp. venerealis and its biotype intermedius and was able to grow, although very weakly, with only carbonic anhydride added to an aerobic atmosphere. The growth obtained using a commercial gas generating microaerophilic kit (Oxoid BR-56) was comparable to the one achieved with the hydrogen rich atmosphere prepared in our laboratory.

Evaluación de drogas antimicrobianas y atmósferas para el aislamiento de Campylobacter fetus subspp. del tracto genital bovino / [Evaluation of antimicrobial drugs and atmospheres for the isolation of Campylobacter fetus subspp. from the bovine genital tract]

Growth of 3 reference bovine C. fetus strains in media with and without antibiotics and bacteriostats active against the most common contaminant bacteria in the bovine genital tract was evaluated. In addition, 2 regional bovine C. fetus strains and 1 reference C. sputorum biovar bubulus strain were used in some experiments. Reference strain C. fetus subsp. venerealis was completely inhibited by polymyxin (> or = 0.25 IU/ml) whereas the other C. fetus strains were not inhibited. In Shepler’s medium supplemented with rifampicin (10 micrograms/ml) subsp. fetus, was the only one to grow. When rifampicin was used at 5 micrograms/ml together with a reduced dose of the other Shepler’s antibiotics, especially polymyxin B (0.85 IU/ml), subsp. venerealis was able to grow; nevertheless even at such a reduced dose, rifampicin was inhibitory for the biotype intermedius. It was demonstrated that triclosan (Irgasan) could be very useful at or = 50 micrograms/ml. C. sputorum biovar. bubulus was less inhibited than C. fetus with triclosan grew up to more than 100 micrograms/ml, with 5-fluorouracil up to 100 micrograms/ml and with fosfomycin up to 50 micrograms/ml. Growth of C. fetus subspp. was compared in different microaerophilic atmospheres contained in anaerobic jars (Oxoid HP 11) without palladium catalyzer. Growth with nitrogen or hydrogen was similar. When jars were replaced by 15 x 13 cm cylindrical cans without valves or gas measurement devices only pure hydrogen supported satisfactory growth of all C. fetus subspp. and C. sputorum biovar. bubulus strains. The candle system, a commercial nitrogen rich gas mixture and pure carbonic anhydride prepared in these cans failed to enable these strains to grow. C. fetus subsp. fetus was more aero-tolerant than subsp. venerealis and its biotype intermedius and was able to grow, although very weakly, with only carbonic anhydride added to an aerobic atmosphere. The growth obtained using a commercial gas generating microaerophilic kit (Oxoid BR-56) was comparable to the one achieved with the hydrogen rich atmosphere prepared in our laboratory.