Tuberculosis-resistant transgenic cattle

Tuberculosis (TB) is a devastating disease that affects humans and many animal species. In humans, TB is mainly caused by Mycobacterium tuberculosis, whereas most cases in cattle are caused by Mycobacterium bovis. However, M. bovis can also cause, albeit rarely, human TB. In PNAS, Wu et al. (1) report the use of novel genetic tools to demonstrate that insertion of one gene into a genomic “safe harbor” can dramatically decrease pathogen replication and disease pathology in M. bovis-infected cattle, and greatly diminish transmission of the disease within cow herds. Could this be a useful approach to diminish TB in cattle and potentially humans?

Tuberculosis and Genetic Control of Resistance

M. bovis has a wide host range and causes significant economic hardship for livestock farmers, with estimates of >50 million cattle infected worldwide. Zoonotic transmission of M. bovis to humans occurs primarily via ingestion of nonpasteurized dairy products or close contact with infected cattle (2). Before wide-scale pasteurization, ∼20–40% of human TB cases resulted from infection with M. bovis (3). The current proportion of human TB cases resulting from M. bovis is low (global median proportion ≤ 1.4%), yet higher rates are reported for Africa (∼2.8%), Mexico (∼7.6%), and Turkey (∼5.3%) (4). Although great strides have been made in the control of bovine tuberculosis in the United States, Australia, New Zealand, and many European countries, this costly and devastating disease persists in other regions, such as the United Kingdom, Republic of Ireland, Africa, China, and Latin America (5). For example, in Great Britain the prevalence of bovine TB has been steadily rising (despite continuous compulsory testing of cattle and targeted culling of the wildlife reservoir, i.e., badgers), with 10% of cattle herds in England (22.7% in the South West Region) under movement restriction and nearly 25,000 cattle slaughtered at a cost of £91 million. The presence of wildlife reservoirs, globalization of economies with increased livestock trade, and limitations on use and efficacy of vaccines severely hinders control of bovine TB. Thus, M. bovis infection in cattle and humans remains a serious problem and continued efforts to decrease its incidence, including novel genetic approaches, are warranted.

There is a modest level of genetic control of susceptibility to adult human TB (6). Variants in the SP110 nuclear body protein gene (SP110), originally identified as the locus (Ipr1) controlling tuberculosis susceptibility in mice (7), have been associated with human TB susceptibility (8, 9), as well as other intracellular bacteria, such as Chlamydia pneumoniae (10).

The work by Wu et al. clearly succeeds as a proof-of-concept that gene transfer can increase resistance to and reduce transmission of M. bovis.

In their study, Wu et al. (1) decided to test the hypothesis that expressing the mouse SP110 gene in bovine macrophages through transgenesis might increase resistance to M. bovis.

Creating Novel Transcription Activator-Like Effector Nucleases to Insert SP110 into a Safe Harbor: Transgenic Cattle Are M. bovis-Resistant

An interesting approach was taken by Wu et al. (1) to maximize probability that the SP110 gene would be expressed correctly. The authors identified a safe harbor for the transgene: an intergenic region in a genome neighborhood containing genes normally expressed in the macrophage, the cell that M. bovis infects and replicates within. The authors theorized that this genome location would remain active even after transgene insertion, thus decreasing the chance of transgene silencing. To insert mouse SP110, they used transcription activator-like effector nucleases (TALENs), which are used to efficiently make random small insertions and deletions (indels) (reviewed in ref. 11). Such indels are caused by the error-prone nonhomologous end-joining repair pathway, after the gene-specific TALEN creates a double-strand break. Thus, the nonhomologous end-joining pathway is not efficient at inserting transgenes of interest; Wu et al. (1) modified their safe harbor TALEN using a mutation known to cause the TALEN to create only a single-strand break. This type of lesion is repaired by the homology-directed repair system, which will “repair” the locus using any supplied homologous sequence. Using their new single-strand break-only TALEN targeting the safe harbor and a DNA fragment with SP110 embedded within the bovine sequences for this region, Wu et al. modified the genome of fibroblasts in culture. Using nuclear transfer, the authors then cloned many calves carrying the SP110 transgene. Macrophages from SP110 transgenic cattle were verified for correct SP110 macrophage expression, and then shown to control M. bovis replication better than that of control cattle, exhibiting a proapoptotic phenotype generally associated with a favorable host outcome (12). Wu et al. (1) then infected transgenic cows by the endobronchial route, mimicking natural infection (5), and compared their responses to those in nontransgenic cows. Bacterial burden and lesion severity was greatly reduced in SP110 transgenic cattle compared with that of control cattle. Thus, although these data demonstrate dissemination of M. bovis beyond the respiratory tract for both genotypes, for at least the first few months postinfection, transgenic cattle seem more resistant to M. bovis replication.

Importantly, Wu et al. (1) did not stop there. The authors also measured whether natural transmission of tuberculosis disease (through cattle to cattle contact) was affected by the transgene. SP110-transgenic and nontransgenic cattle were raised with M. bovis-infected cattle for 12 wk. Transgenic cattle had much lower immunologic responses to the environmental M. bovis, and postmortem examination showed much lower pathology scores in the transgenics than the control cows. Finally, Wu et al. show that macrophages from several offspring of transgenic founders continue to express mouse SP110, and have the same phenotype of resistance to M. bovis infection as seen in their mother’s macrophages. These results indicate that silencing has not occurred in offspring, further validating Wu et al.’s safe harbor approach.

Thus, insertion of SP110 into the cattle genome results in partial, but not complete, resistance to M. bovis infection, similar to what is detected in African Zebu breeds of cattle compared with European breeds, such as Holstein-Friesians (13). Importantly, partial protection to experimental M. bovis challenge may be quite significant. Vaccine efficacy studies have clearly demonstrated that partial protection in experimental infection trials equates to almost complete (95%) protection in field studies (14).

How Does the SP110 Transgene Increase Resistance in Cattle?

Expression of an Ipr1 (SP110) transgene in susceptible mice improved their resistance to M. tuberculosis as well as Listeria (7), and with the results of Wu et al. (1), implies a correlation between SP110 expression and TB resistance. However, higher expression of SP110 mRNA was found in patients with active or latent TB compared with naive groups (15). Wu et al. (1) attribute the increased M. bovis resistance to transgenic macrophage apoptosis compared with necrosis observed for normal macrophages. Esquivel-Solís et al. (16) suggested that a higher level of nitric oxide production was more associated with superior control of M. bovis replication in resistant macrophages, although they did find slightly higher apoptosis as well. More research is clearly required to elucidate the mechanism behind the transgene’s effect, including comparisons of the levels of apoptosis in tissues of infected cattle.

Future Considerations

The work by Wu et al. (1) clearly succeeds as a proof-of-concept that gene transfer can increase resistance to and reduce transmission of M. bovis. Another notable component of this research is the technological advance of insertion of genes into safe harbors in the bovine genome. We note that an important component of the success described here was the availability of genomic knowledge to effect this desired genetic manipulation. Recognizing the importance of genome knowledge for such research, as well as many other aspects of genetics, genome scientists have recently announced a new initiative to greatly expand functional genomics and epigenomics information on domesticated animals (Functional Annotation of Animal Genomes, www.faang.org) (17).

Although the scientific advance here is clear, acceptance of transgenic livestock in the food chain is an open question. Alternatives to transgenetic modification of livestock to improve resistance to TB exist. For example, there is evidence for genetic control of resistance or susceptibility to M. bovis in cattle (18). Recently, several genetic loci were shown to be associated with resistance/susceptibility to M. bovis, none of which were close to the location of the bovine SP110 gene (19). Thus, breeding for resistance to TB may be possible, although pitfalls associated with selection of cattle based on genetic resistance to specific diseases include the potential for increased susceptibility to other diseases (e.g., selection for resistance to M. bovis may result in increased susceptibility to viral and parasitic diseases), increased frequency of heritable diseases, and reduced heterogeneity of immune repertoires. The approach of Wu et al. (1) can make rapid progress, and could circumvent many of these issues because the transgene could be inserted into divergent progenitor cattle. Additionally, the SP110-expressing cattle may in fact be resistant to other intracellular pathogens, as shown in the mouse (7, 10). It would be of great interest to determine resistance to other pathogens for these transgenic cattle, as well as their durability in field environments.

Complete eradication of bovine TB with current methods, including vaccination, may be difficult because of increasing interregional movement of cattle and existing wildlife reservoirs (5). Thus, application of genetic methods for disease resistance (including use of transgenic cattle), along with traditional approaches such as vaccination and test-and-cull campaigns of cattle, as well as efforts to control the disease in wildlife reservoirs via targeted culling and vaccination, may tip the balance in favor of control/eradication of this costly zoonotic disease.