Iron is a fundamental element required by most organisms, including Brucella. Several researchers have suggested that the iron response regulator (irr) and rhizobial iron regulator (rirA) genes regulate iron acquisition by Brucella abortus, influencing heme synthesis by and virulence of this pathogen. However, little is known about another Brucella species, Brucella melitensis. In this research, we successfully constructed two mutants: M5-90Δirr and M5-90ΔrirA. The adhesion, invasion, and intracellular survivability of these two mutants were evaluated in RAW264.7 cells infected with 1 × 106 CFU of M5-90Δirr, M5-90ΔrirA, or M5-90. We also tested the sensitivity of cells to hydrogen peroxide and their ability to grow. In addition, the virulence of these two mutants was evaluated in BALB/c mice. The results showed that the ability of these two mutants to invade and adhere inside the murine macrophages RAW264.7 was attenuated but their ability to replicate intracellularly was strengthened, enhancing the resistance to hydrogen peroxide. The M5-90Δirr mutant showed stronger growth ability than the parental strain under iron-limiting conditions. No differences were observed in the number of bacteria in spleen between M5-90 and M5-90Δirr at 7 or 15 days postinfection. However, the number of M5-90ΔrirA in spleen reduced significantly at 15 days postinfection. The splenic index of the M5-90Δirr group is evidently lower than that of M5-90. This is the first report that irr and rirA genes of B. melitensis are associated not only with virulence but also with growth ability. Together, our data suggest that M5-90Δirr is a promising Brucella vaccine candidate.

We studied livestock abortion and various associated risk factors in the Ili region of northwest China. Livestock abortion prevalence was estimated and correlated with infections (Brucellosis, Salmonellosis, Mycoplasma and Chlamydia seropositivity) and management (farming type and contact with other herds/flocks) risk factors. A total of 2996 serum samples (1406 cow, 1590 sheep) were identified by RBPT (Rose Bengal Plate Test) and c-ELISA (competitive-enzyme linked immunosorbent assay), and they showed the overall seroprevalence of brucellosis in the study area was cow 6.76%, sheep 9.50%. The seroprevalence of brucellosis in X county was cow 7.06%, sheep 9.12%; in H county was cow 11.70%, sheep 10.80%; and in Q county was cow 4.22%, sheep 9.11%. The overall seroprevalence of Mycoplasma in the study area was cow 3.20%, sheep 6.42%. The seroprevalence of Mycoplasma in X county was cow 3.39%, sheep 7.98%; in H county was cow 5.26%, sheep 9.97%; and in Q county was cow 2.11%, sheep 4.33%. The Odds ratio of brucellosis for cow and sheep, respectively, were 45.909 [95% CI 26.912–78.317, P <0.001] and 70.507 [95% CI 43.783–113.544, P <0.001] times higher than other abortion-related factors including mixed farming, contact with other flocks and Mycoplasma infection. A total of 54 samples, including aborted cow (22), sheep (30) fetuses and milk samples (2), were identified as Brucella melitensis ( B . melitensis ) positive. A total of 38 Brucella were isolated from 16 aborted cow, 20 sheep fetuses and 2 milk samples. All of these isolates were identified, and confirmed, as B . melitensis . A phylogenetic tree showed that the Brucella isolates closely matched the B . melitensis biovar 3 isolated in Inner Mongolia, China, and B . melitensis isolated from Norway and India. These results suggest that B . melitensis biovar 3 is the main pathogen responsible for cow and sheep abortion and also pose a human health risk. Additionally, livestock reproduction can also be influenced by Mycoplasma infection and managerial factors (farming type and contact with other herds/flocks), especially in remote areas.

Brucella abortus are Gram-negative and facultative intracellular pathogens affecting humans and animals and can enter various cell types during infection [1]. Brucella penetrates the nasal cavity, oral cavity, and/or pharynx mucous membrane and is phagocytosed by host macrophages, in which they can survive and replicate.

12 We studied livestock abortion and various associated risk factors in the Ili region of 13 northwest China. Livestock abortion prevalence was estimated and correlated with 14 infections (Brucellosis, Salmonellosis, Mycoplasma and Chlamydia seropositivity) and 15 management (farming type and contact with other herds/flocks) risk factors. The 16 prevalence of cow and sheep abortion induced by Brucella was 76.8% (P<0.0001) and 17 84.1% (P<0.0001), and Mycoplasma caused an estimated 15.5% (P=0.025) and 17.6% 18 (P<0.001) abortions, respectively. Abortion-related risk factors included mixed 19 farming (cow P=0.001, sheep P<0.001), contact with other flocks (cow P=0.007, sheep 20 P=0.003), brucellosis positivity (cow P<0.001, sheep P<0.001) and Mycoplasma 21 positivity (cow P=0.031, sheep P<0.001). A total of 2996 serum samples (1402 cow, 22 1594 sheep) were identified by RBPT (Rose Bengal Plate Test), and they showed the 23 seroprevalence of brucellosis in X county was cow 7.1%, sheep 9.1%; in H county was 24 cow 11.7%, sheep 10.7%; and in Q county was cow 4.2%, sheep 9.1%. The 25 seroprevalence of Mycoplasma in X county was cow 3.4%, sheep 7.9%; in H county 26 was cow 5.3%, sheep 9.9%; and in Q county was cow 2.1%, sheep 4.3%. A total of 54 27 samples, including aborted cow (22), sheep (30) fetuses and milk samples (2), were 28 identified as Brucella melitensis (B. melitensis) positive. A total of 38 Brucella were 29 isolated from 16 aborted cow, 20 sheep fetuses and 2 milk samples. All of these isolates 30 were identified, and confirmed, as B. melitensis. A phylogenetic tree showed that the 31 Brucella isolates closely matched the B. melitensis biovar 3 isolated in Inner Mongolia, 32 China, and B. melitensis isolated from Norway and India. These results suggest that B.33 melitensis biovar 3 is the main pathogen responsible for cow and sheep abortion and 34 also pose a human health risk. Additionally, livestock reproduction can also be 35 influenced by Mycoplasma infection and managerial factors (farming type and contact 36 with other herds/flocks), especially in remote areas. 37 38 Introduction 39 Ruminants are a major source of meat production in China and are important for food 40 security. Xinjiang Uygur Autonomous Region (XUAR) is located in northwest China 41 and is a major ruminant production province. In 2017, beef production (0.43 million 42 tons) and mutton production (0.58 million tons) in Xinjiang, respectively, accounted 43 for the 6.8% and 12.4% in the total beef and mutton production in China. Ili is located 44 in the western part of XUAR, where the economy is highly dependent on animal 45 production [1, 2]. The combined number of cattle and sheep is approximately 5.76 46 million in this region. The sheep and cattle are reared under traditional systems, and 47 confined sheep or cattle ranches are the two main feeding systems.48Diseases and poor animal health are major risk factors for animal production in Ili.49 The viability of sheep and cattle production is largely determined by their reproductive ...

Brucella is an intracellular pathogen that invades a host and settles in its immune cells; however, the mechanism of its intracellular survival is unclear. Modification of small ubiquitin-related modifier (SUMO) occurs in many cellular activities. E2 conjugating enzyme 9 (Ubc9) is the only reported ubiquitin-conjugating enzyme that links the SUMO molecule with a target protein. Brucella's intracellular survival mechanism has not been studied with respect to SUMO-related proteins and Ubc9. Therefore, to investigate the relationship between Brucella melitensis 16M and SUMO, we constructed plasmids and cells lines suitable for overexpression and knockdown of SUMO1 and Ubc9 genes. Brucella 16M activated SUMO1/Ubc9 expression in a time-dependent manner, and Brucella 16M intracellular survival was inhibited by SUMO1/Ubc9 overexpression and promoted by SUMO1/Ubc9 depletion. In macrophages, Brucella 16M-dependent apoptosis and immune factors were induced by SUMO1/Ubc9 overexpression and restricted by SUMO1/Ubc9 depletion. We noted no effect on the expressions of SUMO1 and Ubc9 in B. melitensis 16M lipopolysaccharide-prestimulated mouse RAW264.7 macrophages. Additionally, intracellular survival of the 16M△VirB2 mutant was lower than that of Brucella 16M (p < 0.05). VirB2 can affect expression levels of Ubc9, thereby increasing intracellular survival of Brucella in macrophages at the late stage of infection. Collectively, our results demonstrate that B. melitensis 16M may use the VirB IV secretion system of Brucella to interact with SUMO-related proteins during infection of host cells, which interferes with SUMO function and promotes pathogen survival in host cells.