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Silymarin (Sm), the main bioactive principle of Silybum marianum (L.) Gaernt. fruits, has strong antihepatotoxic properties and also exhibits antitumor and antiviral activity. This flavonolignan complex is synthesized from the flavonoid precursor taxifolin, which undergoes an oxidative coupling with the monolignol coniferyl alcohol. Attempts to produce Sm in plant cell suspensions have met with limited success, probably due to insufficient flux into the flavonoid branch pathway of Sm biosynthesis. In this study, it was attempted to increase Sm production in in vitro cultures of S. marianum by genetically engineering the flavonoid pathway. Using Agrobacterium tumefaciens strain C58C1, S. marianum cell suspension cultures were transformed either with the Arabidopsis thaliana transcription factor AtMYB12 (V-myb myeloblastosis viral oncogene homolog) (MYB) or with a chalcone synthase (CHS) gene from Cicer arietinum. A double transformant was also generated by introducing the CaCHS gene in AtMYB12-expressing cultures. The ectopic expression of AtMYB12 in cultures activated some of the genes involved in the flavonoid pathway, notably chalcone isomerase (CHI) and flavonoid 3′-hydroxylase (F3’H), whereas the secondary metabolites altered by the inserted gene were chlorogenic acid glycoside derivatives. Expression of CaCHS in the cultures led to the production of the flavanone naringenin (Ng), which accumulated as glycosides. The simultaneous expression of AtMYB12-CaCHS resulted in the production of lower amounts of chlorogenic derivatives but similar levels of Ng glycosides compared with the CaCHS lines. Both CaCHS lines and the double transformant could be maintained over prolonged subcultures without Ng production losses, confirming long-term and stable CaCHS transgene expression. In no case did the inserted genes have a discernable effect on Sm production compared with the untransformed cultures, neither under control culture conditions or in cultures elicited with MeJA.

Interpreting in vitro bioassays used to determine resistance against acaricides in Rhipicephalus microplus can be challenging without parallel in vivo studies that assess for lost efficacy. The larval immersion test (LIT) is currently the most widely used bioassay to detect ivermectin resistance. The objective of this study was to compare results of the LIT and a field trial using ivermectin in naturally infested cattle. Criteria to consider ticks as resistant with the LIT were based on discriminating doses (DD) and the ratio of lethal concentration (LC) in test populations over the LC of the susceptible Deutch strain, known as the resistance ratio (RR). Ticks were collected from 4 farms, two where ivermectin provided good control of tick infestations and two that claimed lack of efficacy. In two farms where administration of a long-acting ivermectin formulation reduced body tick counts to 45 and 25% of the initial counts at 10-days post-treatment, the RR50 and RR99 were approximately 6 and 20, respectively. The LC50 value approximated the DD for the two farms with claimed resistance, suggesting that about half of the population in each farm was resistant. These LIT values are equal to those reported for the most resistant ticks, which supports the use of the LIT to predict control failure in field situations. The two farms where ivermectin provided good control of tick infestations had LC50s similar to the susceptible strain, although for one farm the LC99 and RR99 suggested incipient resistance.

This study reports the performance of the single intradermal tuberculin (SIT) test and the interferon-gamma (IFN-γ) assay for Mycobacterium bovis in a cattle herd with high prevalence of paratuberculosis (PTB). A total of 58/350 animals were selected for necropsy based on one or more of the following criteria: positive to SIT, IFN-γ, a breeding cow that seroconverted to PTB and showed signs compatible with a wasting disease. Infection status was determined by post mortem diagnostic tests that included histopathology examination, mycobacterial cultures and PCR identification for M. bovis and Mycobacterium avium subsp. paratuberculosis (MAP). In 7/58 animals primary tuberculosis (TB) lesions, affecting only the retropharyngeal and/or mediastinal lymph nodes, were found; 3/7 animals were found SIT positive. PTB was confirmed in 35/58 animals, of which 30 had seroconverted and 14 had typical clinical signs. 45/58 animals were IFN-γ + using the most stringent criterion (cut-off point ≥ 0.05); however, IFN-γ test was only positive in 33 animals when using a higher threshold (cut-off point ≥ 0.1). Three animals co-infected also showed extensive TB and diffuse PTB lesions. These results show that the combined use of SIT and IFN-γ, as interpreted using official guidelines, detected all confirmed cases of TB. Individually, the sensitivity of the SIT was inadequate to diagnose TB-positive animals with an advanced stage of PTB. The large number of IFN-γ + animals with no visible TB lesion could be due, in part, to some protection conferred by prior infection with MAP.

We studied the evolution of different forms of tuberculosis in herds which are periodically submitted to the comparative tuberculin intradermal reaction (CTID) test within the Caprine Tuberculosis Eradication Program in the Region of Murcia (Spain). In the study, 135 goats with tuberculosis from different herds were diagnosed by histopathological and immunocytochemical techniques. Most animals (58 of the 135) were in the primary complex of tuberculosis, with few or no acid-fast bacilli (AFB) and mycobacterial antigens. Generalized tuberculosis was present in 31 of the 135 animals, and the numbers of bacilli and positive immunocytochemical particles were higher than in animals with the primary complex. Postprimary phase was observed in 44 of the 135 animals, and the number of bacilli and positive immunocytochemical particles increased in proportion to the extent and gravity of the necrotic foci. Tuberculous pneumonia with a high number of bacilli was only observed in two goats. Extrapulmonary tuberculosis was present with lesions in the intestines (60/135), liver (80/135) and spleen (77/135). This study confirmed that the Caprine Tuberculosis Eradication Program is resulting in fewer animals with postprimary tuberculous processes, and a corresponding increase in the number of primary complex and generalized tuberculosis.