Bovine campylobacteriosis in heifer: pathogenesis study and insights in the conventional and molecular diagnosis in an experimental bovine model and field cases.

Campylobacter fetus spp. is a bacterium associated to reproductive losses in cattle worldwide. It is a venereal infectious disease known as bovine campilobacteriosis, with high impact mainly in countries with extensive production systems. Here, we show pathogenesis and diagnostic methods for Campylobacter fetus detection in cervico-vaginal mucus (CVM) samples from heifers experimentally infected and field cases from herds with low reproductive performance by campylobacteriosis infection. Bacterial culture, direct immunofluorescence test and qPCR were used as diagnostic methods to evaluate detection of C. fetus. In the experimental model 30 Aberdeen Angus and crossbred heifers and 4 Aberdeen Angus bulls for natural mating were assigned to 3 groups experimentally challenged with C. fetus subsp. fetus (Cff), C. fetus subsps venerealis (Cfv) and C. fetus subsp venerealis biovar intermedius (Cfvi), respectively, and a negative control group, all followed for 9 months. Also, field samples of CVM and aborted fetuses were recollected from seven beef cattle farms. Bacteriological culture had the higher C. fetus detection rate in CVM being the most appropriate, followed by qPCR (with commercial extraction DNA kit), direct immunofluorescence test and qPCR (with in-house extraction DNA method), in both, experimental model and field cases. From experimental model after natural mating, 62.5% and 25% heifers got pregnant from Cff and Cfvi groups, respectively, while from Cfv no pregnancy was detected. The strain more frequently detected was Cfvi, followed by Cff and Cfv. Colonization of Cff in female genital tract with high number of carriers and presence in aborted fetuses was evidenced, suggesting a high risk to bovine reproductive health. Bacteriemia was not detected after genital infection. Given the low detection rate of either test, we suggest the use of both, PCR based methods and bacterial culture could result in higher detection rate in farms with endemic campylobacteriosis.

Bovine campylobacteriosis in bulls: insights in the conventional and molecular diagnosis.

Campylobacter fetus is a gram-negative motile bacterium, with two subspecies relevant to cattle health: C. fetus subsp. venerealis (Cfv) and C. fetus subsp. fetus (Cff). Both subspecies are associated with reproductive losses in cattle. In this study, we evaluated the identification of C. fetus for the diagnosis of bovine campylobacteriosis through bacteriological culture, direct immunofluorescence (DIF) and molecular tests in preputial smegma (PS) samples of three Angus bulls challenged with Cfv, Cfv biovar intermedius (Cfvi) or Cff, respectively, in an experiment imitating the natural infection. Two DNA extraction protocols were tested (in-house thermal extraction and commercial kit). Aspiration and scraping collection for PS were compared by conventional tests. Additionally, bacteremia was also evaluated in blood samples. Bulls were challenged by natural mating with heifers that had been experimentally infected with C. fetus subspecies; which led to infection. The Cfv- and Cfvi-bulls were positive for at least 9 months. Although Cff is not considered a venereal strain, in this study it was transmissible to bull from heifers experimentally infected, as evidenced by its colonization and persistence in the preputial cavity for 5 to 6 months. This finding suggests a potential risk of dissemination within herds. The results obtained by bacteriological culture or direct immunofluorescence (DIF) showed no significant differences, regardless the sampling device used (aspiration with Cassou pipette, metal and plastic scraper). C. fetus qPCR, on the other hand, yielded better results with an in-house DNA extraction method than with a commercial kit (75% vs 66.6%). Furthermore, qPCR diagnosis was more efficient than culture (66.6%) or DIF (56%). Bacteremia in whole blood samples was negative by qPCR and bacteriological culture in all samples. Altogether, this study demonstrated the transmission of Cff from heifers to bull and also showed that PCR-based methods are promising for the diagnosis of Bovine Genital Campylobacteriosis from clinical samples of PS.

Isolation of Campylobacter fetus subsp. venerealis from seminal vesicle of a naturally challenged bull.

Campylobacter fetus is a well-recognized pathogen that affects reproductive rate in cattle. In the present study, two Angus bulls were kept (39 days) separately with a group of heifers experimentally infected with Campylobacter fetus subsp. venerealis (Cfv) and Campylobacter fetus subsp. venerealis biovar intermedius (Cfvi), respectively. Each bull resulted infected post-mating by its respective strain (Cfv and Cfvi). Semen samples collected from each bull at days 39, 82, 132 and 269 resulted positive for C. fetus by bacteriological culture and/or direct immunofluorescence (DIF) test, and confirmed by polymerase chain reaction (PCR) from colonies isolated. Diagnosis resulted better with bacteriological culture (100%) compared to DIF (37,5%). Campylobacter fetus was isolated from seminal vesicle and preputial mucosa by bacteriological culture and confirmed by PCR and DIF test from colonies previously isolated from these tissues (day 276). Microscopic lesions detected in both bulls showed moderate diffuse subepithelial lymphoplasmacytic postitis. None of the seminal vesicle presented relevant microscopic lesions. To our knowledge this is the first report of isolation of C. fetus from seminal vesicles in a bull. The experimental model herein described, mimicks the natural infection and constitutes a promising alternative for future studies of campylobacteriosis in cattle.

L-cysteine transporter-PCR to detect hydrogen sulfide-producing Campylobacter fetus.

Phenotypic differences between Campylobacter fetus fetus and C. fetus venerealis subspecies allow the differential diagnosis of bovine genital campylobacteriosis. The hydrogen sulfide production, for example, is a trait exclusive to C. fetus fetus and C. fetus venerealis biovar intermedius. This gas that can be biochemically tested can be produced from L-cysteine (L-Cys). Herein, we report a novel multiplex-PCR to differentiate C. fetus based on the evaluation of a deletion of an ATP-binding cassette-type L-Cys transporter that could be involved in hydrogen sulfide production, as previously described. A wet lab approach combined with an in silico whole genome data analysis showed complete agreement between this L-Cys transporter-PCR and the hydrogen sulfide production biochemical test. This multiplex-PCR may complement the tests currently employed for the differential diagnosis of C. fetus.

Diversidad de especies de micobacterias no tuberculosas aisladas en ambientes acuáticos de la ciudad de General Pico, La Pampa, Argentina / Species diversity of non-tuberculous mycobacteria isolated from aquatic environments of General Pico city, Province of La Pampa (Argentina)

Las micobacterias no tuberculosas (MNT) no solo se estudian por su importancia como patógenos oportunistas, sino también por sus aplicaciones en biotecnología y biorremediación. Nuestro objetivo fue determinar la presencia de micobacterias en los distintos hábitats acuáticos de la ciudad de General Pico (provincia de La Pampa), así como su diversidad. Los porcentajes de muestras positivas a micobacterias fueron los siguientes: 37,5% en el sistema de distribución de agua de red, 32,6% en el acuífero que abastece dicho sistema, 36,8% en el agua proveniente de las precipitaciones, 53,1% en los humedales del área de influencia, 80% en los natatorios cubiertos y 33,3% en las fuentes decorativas ubicadas en plazas públicas. De los 90 aislamientos de MNT obtenidos el 8,9% no logró ser identificado a nivel de especie con los métodos utilizados, que incluyeron pruebas fenotípicas y métodos moleculares. Las especies más frecuentemente aisladas fueron Mycobacterium fortuitum y Mycobacterium gordonae. Algunas especies identificadas han sido reportadas en casos de micobacteriosis en nuestro país, entre ellas M. fortuitum, M. gordonae, M. intracellulare, M. vaccae, M. lentiflavum y M. nonchromogenicum. No se aislaron MNT en muestras de agua de red con concentraciones de cloro activo residual mayores de 0,8mg/l, mientras que en los natatorios la presencia de hasta 1,5mg/l de cloro activo residual no fue una limitante para la proliferación de estos microorganismos. Se puede considerar que la incidencia de micobacterias en los ambientes acuáticos de General Pico es cercana al 35%, y que la presencia de estos microorganismos y su diversidad se ve afectada por el contacto con el hombre y sus actividades, como así también por la existencia de vida animal.

Non-tuberculous mycobacteria (NTM) are studied not only for their importance as emerging opportunistic pathogens but also for their applications in biotechnology and bioremediation. Our aim was to determine the occurrence and diversity of mycobacteria in different aquatic habitats of General Pico city, Province of La Pampa. The percentage of samples with positive cultures for mycobacteria were the following: 37.5% recovered from the water supply distribution system; 32.6% from the aquifer that supplies water to the distribution system; 36.8% from rain water; 53.1% from the two wetlands in the area of influence; 80% from indoor swimming pools; and 33.3% from water fountains in downtown public squares. Of the 90 NTM isolates, 8.9% could not be identified at the species level with any of the used methods, phenotypic tests and molecular methods. Mycobacterium fortuitum and Mycobacterium gordonae were the most frequently isolated species. Some of the identified species such as, M. fortuitum, M. gordonae, M. intracellulare, M. vaccae, M. lentiflavum and M. nonchromogenicum, have been reported in cases of mycobacteriosis in Argentina. Mycobacteria with values higher than 0.8mg/ml of residual active chlorine were not recovered from the drinking water supply network, whereas in the swimming pools the presence of up to 1.5 mg/l was not a constraint. Based on our results, the presence of mycobacteria in aquatic environments is close to 35% and their occurrence and diversity is affected both by contact with man and his activities as well as by the existence of animal life.

[Species diversity of non-tuberculous mycobacteria isolated from aquatic environments of General Pico city, Province of La Pampa (Argentina)]. / Diversidad de especies de micobacterias no tuberculosas aisladas en ambientes acuáticos de la ciudad de General Pico, La Pampa, Argentina.

Non-tuberculous mycobacteria (NTM) are studied not only for their importance as emerging opportunistic pathogens but also for their applications in biotechnology and bioremediation. Our aim was to determine the occurrence and diversity of mycobacteria in different aquatic habitats of General Pico city, Province of La Pampa. The percentage of samples with positive cultures for mycobacteria were the following: 37.5% recovered from the water supply distribution system; 32.6% from the aquifer that supplies water to the distribution system; 36.8% from rain water; 53.1% from the two wetlands in the area of influence; 80% from indoor swimming pools; and 33.3% from water fountains in downtown public squares. Of the 90 NTM isolates, 8.9% could not be identified at the species level with any of the used methods, phenotypic tests and molecular methods. Mycobacterium fortuitum and Mycobacterium gordonae were the most frequently isolated species. Some of the identified species such as, M. fortuitum, M. gordonae, M. intracellulare, M. vaccae, M. lentiflavum and M. nonchromogenicum, have been reported in cases of mycobacteriosis in Argentina. Mycobacteria with values higher than 0.8mg/ml of residual active chlorine were not recovered from the drinking water supply network, whereas in the swimming pools the presence of up to 1.5mg/l was not a constraint. Based on our results, the presence of mycobacteria in aquatic environments is close to 35% and their occurrence and diversity is affected both by contact with man and his activities as well as by the existence of animal life.

A Closed-tube Loop-Mediated Isothermal Amplification Assay for the Visual Endpoint Detection of Brucella spp. and Mycobacterium avium subsp. paratuberculosis.

LAMP (loop-mediated isothermal amplification) is an isothermal nucleic acid amplification technique that is characterized by its efficiency, rapidity, high yield of final product, robustness, sensitivity, and specificity, with the blueprint that it can be implemented in laboratories of low technological complexity. Despite the conceptual complexity underlying the mechanistic basis for the nucleic acid amplification, the technique is simple to use and the amplification and detection can be carried out in just one step. In this chapter, we present a protocol based on LAMP for the rapid identification of isolates of Brucella spp. and Mycobacterium avium subsp. paratuberculosis, two major bacterial pathogens in veterinary medicine.

LAMP technology: Rapid identification of Brucella and Mycobacterium avium subsp. paratuberculosis.

In this study, we developed new sets of primers to detect Brucella spp. and M. avium subsp. paratuberculosis (MAP) through isothermal amplification. We selected a previously well-characterized target gene, bscp31, specific for Brucella spp. and IS900 for MAP. The limits of detection using the loop-mediated isothermal amplification (LAMP) protocols described herein were similar to those of conventional PCR targeting the same sequences. Hydroxynaphtol blue and SYBR Green(TM) allowed direct naked-eye detection with identical sensitivity as agarose gel electrophoresis. We included the LAMP-based protocol in a rapid identification scheme of the respective pathogens, and all tested isolates were correctly identified within 2 to 3 h. In addition, both protocols were suitable for specifically identifying the respective pathogens; in the case of Brucella, it also allowed the identification of all the biovars tested. We conclude that LAMP is a suitable rapid molecular typing tool that could help to shorten the time required to identify insidious bacteria in low-complexity laboratories, mainly in developing countries.

LAMP technology: Rapid identification of Brucella and Mycobacterium avium subsp. paratuberculosis

In this study, we developed new sets of primers to detect Brucella spp. and M. avium subsp. paratuberculosis (MAP) through isothermal amplification. We selected a previously well-characterized target gene, bscp31, specific for Brucella spp. and IS900 for MAP. The limits of detection using the loop-mediated isothermal amplification (LAMP) protocols described herein were similar to those of conventional PCR targeting the same sequences. Hydroxynaphtol blue and SYBR GreenTM allowed direct naked-eye detection with identical sensitivity as agarose gel electrophoresis. We included the LAMP-based protocol in a rapid identification scheme of the respective pathogens, and all tested isolates were correctly identified within 2 to 3 h. In addition, both protocols were suitable for specifically identifying the respective pathogens; in the case of Brucella, it also allowed the identification of all the biovars tested. We conclude that LAMP is a suitable rapid molecular typing tool that could help to shorten the time required to identify insidious bacteria in low-complexity laboratories, mainly in developing countries.

In vivo short-term exposure to residual oil fly ash impairs pulmonary innate immune response against environmental mycobacterium infection.

Epidemiological studies have shown that pollution derived from industrial and vehicular transportation induces adverse health effects causing broad ambient respiratory diseases. Therefore, air pollution should be taken into account when microbial diseases are evaluated. Environmental mycobacteria (EM) are opportunist pathogens that can affect a variety of immune compromised patients, which impacts significantly on human morbidity and mortality. The aim of this study was to evaluate the effect of residual oil fly ash (ROFA) pre-exposure on the pulmonary response after challenge with opportunistic mycobacteria by means of an acute short-term in vivo experimental animal model. We exposed BALB/c mice to ROFA and observed a significant reduction on bacterial clearance at 24 h post infection. To study the basis of this impaired response four groups of animals were instilled with (a) saline solution (Control), (b) ROFA (1 mg kg(-1) BW), (c) ROFA and EM-infected (Mycobacterium phlei, 8 × 10(6) CFU), and (d) EM-infected. Animals were sacrificed 24 h postinfection and biomarkers of lung injury and proinflammatory madiators were examined in the bronchoalveolar lavage. Our results indicate that ROFA was able to produce an acute pulmonary injury characterized by an increase in bronchoalveolar polymorphonuclear (PMN) cells influx and a rise in O2 (-) generation. Exposure to ROFA before M. phlei infection reduced total cell number and caused a significant decline in PMN cells recruitment (p < 0.05), O2 (-) generation, TNFα (p < 0.001), and IL-6 (p < 0.001) levels. Hence, our results suggest that, in this animal model, the acute short-term pre-exposure to ROFA reduces early lung response to EM infection.

Low levels of residual oil fly ash (ROFA) impair innate immune response against environmental mycobacteria infection in vitro.

Epidemiological studies have shown that pollution derived from industrial and vehicular transportation provokes adverse health effects causing broad spectrum of ambient respiratory diseases. Therefore, air pollution should be taken into account when microbial diseases are evaluated. Environmental mycobacteria (EM) are opportunist pathogens in a variety of immunocompromised patients eliciting significant impact on human morbidity and mortality. The aim of this study was to evaluate the in vitro effects of residual oil fly ash (ROFA) on the alveolar macrophages (AMs) response to opportunistic bacteria. AMs from young Wistar rats were obtained by bronchoalveolar lavage and co-cultured with Mycobacterium phlei (MOI 10). We exposed AM cultures to ROFA to characterize the effect of low ROFA concentrations (0, 2.5, and 5µg/ml) and evaluated the response of pre-exposed AM against the bacilli. Low ROFA concentrations induced superoxide anion and nitrites production (p<0.001). Pre-exposure to ROFA (2.5 and 5µg/ml) caused a significant reduction on TNFα (p<0.001) and superoxide anion (p<0.001) production but, did not modify the nitrite production when AM were co-cultured with M. phlei. In addition, ROFA significantly diminished AM killing ability in culture (p<0.001). Hence, our results indicate that pre-exposure to low levels of ROFA modifies the innate pulmonary defence mechanisms against environmental mycobacteria.