A neutrophil-based test as an auxiliary tool for substantiating the diagnosis of bovine tuberculosis.

Background: Bovine tuberculosis (bTB) is still a prominent threat to animal health; lacking an efficient vaccine, other than BCG to get rid of tuberculosis, the most effective way for this is culling and slaughtering the infected animals. There are several cellular, serological, and molecular tests for the diagnosis of the disease but the most practical one at the field level is the double skin testing with bovine and aviary tuberculins. This is not a very specific test but is sensitive enough to identify most diseased animals; adjunct practical tests are desirable to strengthen the utility of skin tests. All lymphoid and myeloid cells participate, in diverse grades, in the immune response to tuberculosis with neutrophils playing an unintended pathologic role. The study aimed to investigate the response of neutrophils to agents present in the sera of tuberculous cows. Methods: We have developed a neutrophil-based test (N BT) to identify diseased cows within a herd suspected of having tuberculosis; a positive N BT correlates with a positive double skin test. In this test, healthy neutrophils are incubated with the sera of healthy or tuberculous cows for 3 and 6 h, and the nuclear morphologic changes are recorded and analyzed. Results: Sera from tuberculous but not from healthy cows induce nuclear alterations including pyknosis, swelling, apoptosis, and sometimes NETosis, in healthy neutrophils, and CFP 10 and ESAT 6 participate in the phenomenon. Conclusion: We propose the N BT as an auxiliary tool for substantiating the diagnosis of bTB reinforcing the PPD test outcome to help decide whether or not a cow should be sacrificed.

Detection of Mycobacterium avium subsp. paratuberculosis in reproductive tissue and semen of naturally infected rams.

Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of paratuberculosis (PTB), disease that causes a syndrome of bad nutrient absorption, weight loss and eventually death. The intestine is the main target organ where the infection develops; however, there is evidence of infection by MAP in extra-intestine sites of sheep, including mesenteric nodes and semen. The aim of the study was to identify the presence of MAP in reproductive tissue and semen of infected Pelibuey rams in clinical state of PTB. Seven rams were used in clinical PTB state and a non-infected ram by MAP of the Pelibuey breed, confirmed by serology, nPCR and bacteriological culture, with average weight and age of 57.23 ± 1.73 kg and 2.91 ± 0.17 years, respectively. The presence of MAP was identified in different tissue samples: spleen (1/7, 14.3% and 2/7, 28.6%), small intestine (3/7, 42.9% and 4/7, 57.1%) and mesenteric lymph nodes (3/7, 42.9% and 3/7, 42.9%), with nPCR and culture, respectively. It was also identified in epididymis tissue (1/7, 14.3%), Cowper gland (2/7, 28.6%) and prostate (1/7, 14.3%), using nPCR, although without detection in culture. It was identified in testicular tissue in 42.8% (3/7; culture or nPCR technique), but in 28.6% (2/7) with both techniques. Finally, the presence of MAP was identified in 42.9% (3/7) of semen samples with nPCR; however, it was not detected through culture. In conclusion, the presence of MAP was identified in lymphatic, digestive tissue, and semen; the presence of MAP was reported for the first time in epididymis, Cowper gland, prostate and testicles of infected Pelibuey rams.

Detección de Mycobacterium avium subespecie paratuberculosis, por medio de PCR-anidada a partir de muestras de heces de ovinos / Detection of Mycobacterium avium subspecies paratuberculosis by nested-PCR of ovine fecal samples

Paratuberculosis is a chronic granulomatous enteritis caused by Mycobacterium avium subspecies paratuberculosis (Map), which affects wild and domestic ruminants. Map is shed in feces from infected animals. Transmission of the infection takes place by oral ingestion of the bacterium from contaminated food and water with feces. With the objective to establish a paratuberculosis diagnosis in ovine by nested-PCR from fecal samples, 204 fecal and serum ovine samples were studied. Feces were evaluated by nested-PCR and bacterial culture, serum samples were analyzed by agar gel immunodiffusion (AGID). Nested-PCR yielded a 210 bp amplification product that corresponds to Map-IS900, in 61 out of 204 samples. From these, 43 were from AGID positive animals and 18 from negative animals. Seventeen Map strains were isolated by bacterial culture and AGID detected 91 positive animals. Nested-PCR allowed to detect, sooner, greater number of animals shedding bacillus, even when they had resulted negative to the serological test. This result is considered important because generally these animals, while remaining in the farm, constitute the main source of infection for the herd. Nested-PCR should be considered as an alternative, when a prompt result is required to know the health status of the herd with respect to paratuberculosis.

La paratuberculosis es una enteritis granulomatosa de curso crónico ocasionada por Mycobacterium avium subespecie paratuberculosis (Map), afecta a rumiantes domésticos y silvestres. Map es excretada en las heces de animales que desarrollan la enfermedad, y la transmisión de la infección se da mediante la ingestión de alimentos y agua contaminados por heces de animales infectados. Con el objetivo de establecer el diagnóstico de paratuberculosis en ovinos por medio de la PCR-anidada a partir de muestras de heces, se trabajaron 204 muestras de heces y sueros de ovinos; las heces se evaluaron por PCR-anidada y cultivo bacteriológico, las muestras de sueros fueron analizadas por medio de inmunodifusión en agar gel (lDGA). Con la PCR-anidada se obtuvo un producto de amplificación 210 pb que corresponde a la IS900 de Map, en 61 de las 204 muestras. De éstas, 43 eran de animales positivos a IDGA y 18 negativos. Mediante cultivo bacteriológico se aislaron 17 cepas de Map; en este contexto, la IDGA detectó a 91 animales como positivos. La PCR-anidada permitió detectar en menor tiempo a mayor cantidad de animales que estaban eliminando al bacilo, aun cuando habían resultado negativos a la prueba serológica; este resultado se considera importante, ya que generalmente estos animales, al permanecer dentro de la granja, constituyen la principal fuente de infección para el rebaño. Se debe considerar a la PCR-anidada como alternativa, cuando se requiera el diagnóstico en breve tiempo, para conocer el estado sanitario del rebaño con respecto a paratuberculosis.

Polymorphism of the PE domain of PE/PE_PGRS sequences in clinical isolates of Mycobacterium bovis in Mexico.

Polymorphism of the PE domain of PE/PE_PGRS sequences was studied in Mycobacterium bovis isolates from different Mexican states. Samples were analyzed by spolygotyping and RFLP using IS6110 and a 235-bp fragment of the PE domain of PE/PE_PGRS as probes. With the PE probe, three different genotypes were observed, one being predominant in all states. These results confirm the high conservation of the PE domain and suggests a potential role for PE sequence as a stable genetic marker for bovine tuberculosis.

Aislamiento e Identificación de Mycobacterium Bovis a Partir de Muestras de Expectoración de Pacientes Humanos con Problemas Respiratorios Crónicos / Isolation and identification of Mycobacterium bovis from sputum samples of human patients with chronic respiratories diseases

Se realizó un estudio bacteriológico en 9 muestras con examen de esputo positivo y 41 muestras con examen de esputo negativo, obtenidas de pacientes humanos con problemas respiratorios crónicos, con tos persistente y mala respuesta al tratamiento contra tuberculosis. Las muestras se sembraron por duplicado en los medios de cultivo de Lowenstein-Jensen y Stonebrink. Todas las 9 muestras con examen positivo a ZN fueron también positivas al aislamiento de micobacterias; de las 41 muestras negativas a ZN, 10 (24.0 por ciento) fueron también positivas al aislamiento. Trece de estos crecimientos se identificaron como M.tuberculosis, tres correspondieron a M.bovis y en tres se identificó un crecimiento mixto que correspondió a ambas micobacterias. El hecho de que 24.0 por ciento de las muestras negativas al examen de frotis de esputo resultaran positivas al cultivo remarca la importancia del diagnóstico por aislamiento de la micobacteria. Además del aislamiento, la tipificación bioquímica también es importante ya que esto decidirá el tratamiento apropiado debido a que M.bovis es naturalmente resistente a la pirazinamida