Association of an IRF3 putative functional uORF variant with resistance to Brucella infection: A candidate gene based analysis of InDel polymorphisms in goats.

Brucellosis is an important zoonotic disease caused by infection with Brucella spp. It generates major economic losses in livestock production worldwide. Goats are the principal hosts of B. melitensis, the main infection agent of caprine and human brucellosis. The selection of resistance-related genes is considered one of the best long-term means to improve control to bacterial infection in domestic ruminants. We performed a candidate gene association study to test if six short insertion/deletion polymorphisms (InDels) at bacterial-infection related genes influence the resistance to Brucella infection in female creole goats. InDels (IRF3-540: rs660531540, FKBP5-294: rs448529294, TIRAP-561: rs657494561, PTPRT-588: rs667380588, KALRN-989: rs667660989 and RAB5a-016: rs661537016) were resolved by PCR-capillary electrophoresis in samples from 64 cases and 64 controls for brucellosis. Allelic frequencies were significantly different between cases and controls at IRF3-540 and KALRN-989 (p = 0.001 and 0.005). Indeed, the minor alleles (a and k) at InDels IRF3-540 and KALRN-989 were more frequent among controls than cases, providing evidence that these alleles confer protection against Brucella infection. Moreover, IRF3-540 a-containing genotypes (Aa and aa) were associated with absence of Brucella-specific antibodies in goats (p = 0.003; OR = 3.52; 95% CI = 1.55-7.96), and more specifically, a-allele was associated with resistance to Brucella infection in a dose-dependent manner. Also, we observed that the IRF3-540 deletion (a-allele) extends a conserved upstream ORF by 75 nucleotides to the main ORF, and thus it may decrease gene expression by reducing translation efficiency from the main ORF. These results suggest a potential functional role of IRF3-540 deletion in genetic resistance to Brucella infection in goats.

Cytokine expression profile of B. melitensis-infected goat monocyte-derived macrophages.

Brucella parasitize the macrophage where is able to replicate and modulate the immune response in order to establish a chronic infection. The most adequate response to control and eliminate Brucella infection is a type 1 (Th1) cell-mediated effector immunity. Research in immune response of B. melitensis-infected goats is relatively scarce. In this study, we first evaluated changes in the gene expression of cytokines, a chemokine (CCL2) and the inducible nitric oxide synthase (iNOS) of goat macrophage cultures derived from monocytes (MDMs) infected for 4 and 24 h with Brucella melitensis strain 16 M. TNFα, IL-1ß and iNOS, and IL-12p40, IFNγ and also iNOS were significantly expressed (p < 0.05) at 4 and 24 h respectively, in infected compared to non-infected MDMs. Therefore, the in vitro challenge of goat MDMs with B. melitensis promoted a transcriptional profile consistent with a type 1 response. However, when the immune response to B. melitensis infection was contrasted between MDM cultures phenotypically restrictive or permissive to intracellular multiplication of B. melitensis 16 M, it was observed that the relative IL-4 mRNA expression was significantly higher in permissive macrophage cultures with respect to restrictive cultures (p < 0.05), independently of the time p.i. A similar trend, although non-statistical, was recorded for IL-10, but not for pro-inflammatory cytokines. Thus, the up-expression profile of inhibitory instead of pro-inflammatory cytokines could explain, in part, the difference observed in the ability to restrict intracellular replication of Brucella. In this sense, the present results make a significant contribution to the knowledge of the immune response induced by B. melitensis in macrophages of its preferential host species.

Comparative Review of Brucellosis in Small Domestic Ruminants.

Brucella melitensis and Brucella ovis are the primary etiological agents of brucellosis in small domestic ruminants. B. melitensis was first isolated in 1887 by David Bruce in Malta Island from spleens of four soldiers, while B. ovis was originally isolated in Australia and New Zealand in early 1950's from ovine abortion and rams epididymitis. Today, both agents are distributed worldwide: B. melitensis remains endemic and associated with an extensive negative impact on the productivity of flocks in -some regions, and B. ovis is still present in most sheep-raising regions in the world. Despite being species of the same bacterial genus, B. melitensis and B. ovis have extensive differences in their cultural and biochemical characteristics (smooth vs. rough colonial phases, serum and CO2 dependence for in vitro growth, carbohydrate metabolism), host preference (female goat and sheep vs. rams), the outcome of infection (abortion vs. epididymitis), and their zoonotic potential. Some of these differences can be explained at the bacterial genomic level, but the role of the host genome in promoting or preventing interaction with pathogens is largely unknown. Diagnostic techniques and measures to prevent and control brucellosis in small ruminants vary, with B. melitensis having more available tools for detection and prevention than B. ovis. This review summarizes and analyzes current available information on: (1) the similarities and differences between these two etiological agents of brucellosis in small ruminants, (2) the outcomes after their interaction with different preferred hosts and current diagnostic methodologies, (3) the prevention and control measures, and (4) alerting animal producers about the disease and raise awareness in the research community for future innovative activities.

Evaluation of the safety profile of the vaccine candidate Brucella melitensis 16MΔvjbR strain in goats.

Small ruminant brucellosis is caused by the Gram negative cocci-bacillus Brucella (B.) melitensis, the most virulent Brucella species for humans. In goats and sheep, middle to late-term gestation abortion, stillbirths and the delivery of weak infected offspring are the characteristic clinical signs of the disease. Vaccination with the currently available Rev. 1 vaccine is the best option to prevent and control the disease, although it is far from ideal. In this study, we investigate the safety of the B. melitensis 16MΔvjbR strain during a 15-month period beginning at vaccination of young goats, impregnation, delivery and lactation. Forty, 4 to 6 months old, healthy female crossbreed goats were randomly divided into four groups (n = 10) and immunized subcutaneously with a single vaccine dose containing 1x109 CFU of B. melitensis 16MΔvjbR delivered in alginate microcapsules or non-encapsulated. Controls received empty capsules or the commercially available Rev.1 vaccine. Seven months post-vaccination, when animals were sexually mature, all goats were naturally bred using brucellosis-free males, and allowed to carry pregnancies to term. Blood samples to assess the humoral immune response were collected throughout the study. At two months post-delivery, all dams and their offspring were euthanized and a necropsy was performed to collect samples for bacteriology and histology. Interestingly, none of the animals that received the vaccine candidate regardless of the formulation exhibited any clinical signs associated with vaccination nor shed the vaccine strain through saliva, vagina or the milk. Gross and histopathologic changes in all nannies and offspring were unremarkable with no evidence of tissue colonization or vertical transmission to fetuses. Altogether, these data demonstrate that vaccination with the mutant strain 16MΔvjbR is safe for use in the non-pregnant primary host.

Caprine brucellosis: A historically neglected disease with significant impact on public health.

Caprine brucellosis is a chronic infectious disease caused by the gram-negative cocci-bacillus Brucella melitensis. Middle- to late-term abortion, stillbirths, and the delivery of weak offspring are the characteristic clinical signs of the disease that is associated with an extensive negative impact in a flock's productivity. B. melitensis is also the most virulent Brucella species for humans, responsible for a severely debilitating and disabling illness that results in high morbidity with intermittent fever, chills, sweats, weakness, myalgia, abortion, osteoarticular complications, endocarditis, depression, anorexia, and low mortality. Historical observations indicate that goats have been the hosts of B. melitensis for centuries; but around 1905, the Greek physician Themistokles Zammit was able to build the epidemiological link between "Malta fever" and the consumption of goat milk. While the disease has been successfully managed in most industrialized countries, it remains a significant burden on goat and human health in the Mediterranean region, the Middle East, Central and Southeast Asia (including India and China), sub-Saharan Africa, and certain areas in Latin America, where approximately 3.5 billion people live at risk. In this review, we describe a historical evolution of the disease, highlight the current worldwide distribution, and estimate (by simple formula) the approximate costs of brucellosis outbreaks to meat- and milk-producing farms and the economic losses associated with the disease in humans. Successful control leading to eradication of caprine brucellosis in the developing world will require a coordinated Global One Health approach involving active involvement of human and animal health efforts to enhance public health and improve livestock productivity.