Yongxia Liu

Liposomes containing recombinant E protein vaccine against duck Tembusu virus in ducks

To obtain an effective vaccine candidate against duck Tembusu viral (DTMUV) disease which causes egg-drop and great economical loss in the Chinese duck industry, liposome vaccines containing recombinant E protein were prepared and assessed in this study. The recombinant plasmid (PET28a-E) was constructed and transformed into BL21 (DE3) cells to produce E proteins. The recombinant E proteins were purified and entrapped by liposomes through reverse-phase evaporation. Eighty-four cherry valley ducks were randomly divided into seven groups and inoculated intramuscularly at one- or seven-day-old with liposomes-E protein or Freunds adjuvant-E protein vaccine. Blood samples were collected from the first week to the tenth week for serum antibody, plasma for viremia, as well as oropharyngeal and cloacal swabs for virus shedding analyses after being challenged with a 10(2.4) 50% tissue culture infective dose (TCID50) of duck Tembusu virus. Results showed that serum antibody level of the liposomes vaccine was higher than the Freunds adjuvant vaccine, and inoculating twice was superior to once; furthermore, the viremia and virus shedding tests also proved that the liposomes vaccine can provide complete protection against DTMUV challenge. These results demonstrated that the liposomes-E protein vaccine could be used as a potential candidate vaccine to prevent DTMUV infection in ducks.

Serological survey of canine dirofilariosis in Chongqing, Kunming, Nanchang, Fuzhou, Guangzhou, Shenzhen, and Nanning in Southern China.

The present study conducts a serological survey on the presence of canine dirofilariosis in domestic dogs using an enzyme-linked immunosorbent assay (ELISA) kit. A total of 310 household dogs (166 females and 144 males) in Chongqing, Kunming, Nanchang, Fuzhou, Guangzhou, Shenzhen, and Nanning in Southern China were examined. Of the 310 dogs, 42 (13.5%) were seropositive for dirofilariosis. No statistically significant difference was observed in terms of sex in the seroprevalence of dirofilariosis using the ELISA kit. The positive rates for dirofilariosis were 6.6% in the 0-1-year-old group, 13.8% in the 1-4-year-old group, and 21.6% in the less than 4-year-old group. The statistical analysis revealed that significant differences were observed in the 1-4-year-old group (P=0.037, OR=0.441, 95% CI=0.170-1.144) and less than 4-year-old group (P<0.001, OR=0.256, 95% CI=0.095-0.693). In the regional comparison, the shoreline city Shenzhen (18.8%) had a significantly higher prevalence than urban and mountain areas (P<0.05, OR=0.310, 95% CI=0.066-1.445). In conclusion, Dirofilaria immitis infection in domestic dogs was present in Chongqing, Kunming, Nanchang, Fuzhou, Guangzhou, Shenzhen, and Nanning. Therefore, heartworm treatment and/or chemoprophylaxis for the captured domestic dogs are necessary in these areas. To the best of our knowledge, the present study is the first using serological methods to examine D. immitis infection in domestic dogs in Mainland China in the recent years.

Seroprevalence of Toxoplasma gondii in Dogs in Shandong, Henan, and Heilongjiang Provinces, and in the Xinjiang Uygur Autonomous Region, People's Republic of China

abstract:  Seroprevalence of Toxoplasma gondii infection was determined in sera of 632 dogs (551 pets, 81 strays) from Shandong, Henan, and Heilongjiang Provinces, and in the Xinjiang Uygur Autonomous Region, Peoples Republic of China (PRC), using the indirect hemagglutination assay (cutoff titer 1∶64 or higher); 11.1% were seropositive. The seroprevalence in stray dogs and in ≥3-yr-old dogs was significantly higher (P < 0.05) than that in household dogs and in <3-yr-old dogs. There were no significant differences in terms of gender, breed, or locality (P ≥ 0.05). The results indicate that T. gondii infections are common in dogs in PRC.

gp85 protein vaccine adjuvanted with silica nanoparticles against ALV-J in chickens

This study focused on the effect of silica nanoparticles as adjuvant for vaccine applications comprised of gp85, a dominating structural protein of J Subgroup Avian Leukosis Virus (ALV-J), and which was evaluated by comparing with the responsiveness induced by that emulsified in Freund adjuvant. Thirty-six chickens were inoculated twice with gp85 adjuvanted with the silica nanoparticles or Freunds adjuvant at the 2nd and 3rd week old. Two weeks later, the inoculated chickens were challenged with a 102.2 50% tissue culture infective dose (TCID50) of ALV-J. The blood samples were collected weekly to detect the serum antibodies and viremia. Results showed that positive serum antibodies (S/P value>0.6) against gp85 emerged at the third week in the inoculated chickens, while the antibodies level persisted longer in silica nanoparticles adjuvanted-group to Freunds adjuvanted-group. Furthermore, viremia in silica nanoparticles adjuvanted-group was recovered more quickly compared with Freunds adjuvanted-group. Hence our study revealed that silica nanoparticles can effectively improve the protection of gp85 vaccine against ALV-J and present a better performance than Freunds adjuvant.

Moderate selenium dosing inhibited chromium (VI) toxicity in chicken liver

This study aimed to clarify the effect of selenium (Se) on chromium (VI) [Cr(VI)]‐induced damage in chicken liver. A total of 105 chickens were randomly divided into seven groups of 15. Group I received deionized water; group II received Cr(VI) (7.83 mg/kg/d) alone; and other groups orally received both Cr(VI) (7.83 mg/kg/d) and Se of different doses (0.14, 0.29, 0.57, 1.14, and 2.28 mg/kg/d). The levels of superoxide dismutase (SOD), glutathione (GSH), malondialdehyde (MDA), Ca2+‐ATPase, and mitochondrial membrane potential (MMP) were measured. Results showed that Cr(VI) increased MDA content and decreased GSH content, T‐SOD activity, Ca2+‐ATPase activity, and MMP level. Meanwhile, Se co‐treatment (0.14, 0.29, and 0.57 mg/kg/d) increased the viability of the above indicators compared with Cr(VI)‐treatment alone. In addition, histopathologic examination revealed that Cr(VI) can cause liver damage, whereas Se supplementation of moderate dose inhibited this damage. This study confirmed that Se exerted protective effect against Cr(VI)‐induced liver damage.

Synthesis, cloning, and expression of Mycoplasma suis inorganic pyrophosphatase gene using PCR-based accurate synthesis and overlap-extension PCR, and its immunogenicity analysis

Mycoplasma suis (M. suis), a hemotrophic pathogen of pigs, causes economic losses in swine production throughout the world. Inorganic pyrophosphatase (ppa) is a very important gene in M. suis. The ppa gene of M. suis was synthesized by PCR-based accurate synthesis (PAS) and overlapextension PCR, inserted into vector pMD18-T, and then subcloned to the prokaryotic expression vector pET28c.The recombinant plasmid pET28c_ppa was transformed to E. coli BL21 for expression under induction of isopropyl thiogalactoside. The expressed product was identified by SDS-PAGE and Western blot, which suggested that the recombinant protein has good antigenicity. Piglets were immunised with purified recombinant protein, and specific antibodies to the recombinant protein were detected in piglet serum. The results show that the ppa gene can be efficiently expressed in E. coli and that the expressed recombinant protein can elicit a specific serum antibody response in piglets. PAS and overlap-extension PCR were first used to synthesize the ppa of M. suis. They provide simple, rapid, reliable and relatively inexpensive methods to synthesize, clone, and express genes. The experiment conducted in this paper will enable future research into the role and function of the ppa gene.

Emergence of Morganella morganii subsp. morganii in dairy calves, China

Morganella morganii (M. morganii), a gram-negative bacterium, is found in the intestinal tracts of humans and in the environment. In 1906, M. morganii was first isolated from a pediatric fecal culture and identified as an unimportant pathogen. Since the 1970s, M. morganii has been considered an important cause of nosocomial infection, and some infections lead to a high mortality rate. The types of diseases associated with M. morganii infection vary and include cellulitis, abscessation, sepsis, diarrhea, and bacteremia. To date, M. morganii infection has been reported in various animals including reptiles, elephant seals, broiler chickens, piglets, jaguars, guinea pigs, rabbits, and dolphins. Notably, M. morganii can exist in an animal’s oral cavity and cause human infection if an animal bites or scratches a human; thus, M. morganii has evolved as a zoonotic pathogenic bacteria. However, there is no evidence that M. morganii causes disease in cattle. In the present study, we identified M. morganii-infected cattle for the first time.M. morganii infection resulted in a high mortality rate (57%) and severe pathological lesions. Moreover, our survey showed that M. morganii originated from the milk of imported cattle. The diseased cattle were from a farm located in Tai’an, Shandong, China. The cattle farm owned 200 cattle and 100 calves, all of which were Holstein cows. In November 2017, the newborn calves showed signs of depression, poor appetite, and paralysis and excreted egg white-like feces with white flocculant material (Fig. 1a). Five days after the onset of sickness, eight of fourteen (57%) calves died, and the surviving calves showed emaciation and growth retardation. Antibiotics, such as gentamycin, sulfadiazine, penicillin, and florfenicol, were used to treat the sick cows. However, none of them were effective for treatment. Correspondingly, we conducted drug sensitivity tests of 37 drugs with the Kirby–Bauer disc diffusion method according to the National Committee for Clinical Laboratory Standards (NCCLS) regulations. The results showed that M. morganii was resistant to sulfadiazine, penicillin, and florfenicol and had intermediate sensitivity to gentamicin (Supplementary Table 1). These results indicated that the isolated M. morganii was resistant to multiple antibiotics in cattle. The autopsy results showed a large number of light yellow fibrinous suppurative clots in the abdominal cavity (Fig. 1b), gastric contents outflowing from an ulcer in the gastric fundus (Fig. 1c), white fibrinous protein on the surface of the spleen (Fig. 1d), a large amount of yellow pericardial effusion and white septic exudate in the epicardium (Fig. 1e), liver abscessation with white fibrinous purulent exudate on the surface (Fig. 1f), and white purulent material in the bladder (Fig. 1g). The lungs had connective tissue hyperplasia in the anterior lobe, edema in the posterior lobe, and a large amount of white foamy edema fluid in the trachea (Fig. 1h). The kidneys had several yellow suppurative nodules in the renal papillae (Fig. 1i). To identify which bacteria induced the infection, we sampled specimens of the infected heart, liver, spleen, lung, and kidney with an inoculation ring to inoculate tryptic soy agar (TSA), potato dextrose agar (PDA), and blood agar in an aseptic environment. Colonies grew in these three media and were gray, smooth, and round. Five colonies were selected to inoculate vegetative broth at 37 ° C for 12 h with agitation, and then the bacterial DNA was extracted using the Mericon DNA Bacteria Plus Kit (Qiagen, Hilden, Germany). The bacterial 16S rDNA universal primers were used for PCR. After cloning,

Analysis of molecular evolution of nucleocapsid protein in Newcastle disease virus

The present study investigated the molecular evolution of nucleocapsid protein (NP) in different Newcastle disease virus (NDV) genotypes. The evolutionary timescale and rate were estimated using the Bayesian Markov chain Monte Carlo (MCMC) method. The p-distance, Bayesian skyline plot (BSP), and positively selected sites were also analyzed. The MCMC tree indicated that NDV diverged about 250 years ago with a rapid evolution rate (1.059 × 10−2 substitutions/site/year) and that different NDV genotypes formed three lineages. The p-distance results reflected the great genetic diversity of NDV. BSP analysis suggested that the effective population size of NDV has been increasing since 2000 and that the basic reproductive number (R0) of NDV ranged from 1.003 to 1.006. The abundance of negatively selected sites in the NP and the mean dN/dS value of 0.07 indicated that the NP of NDV may have undergone purifying selection. However, the predicted positively selected site at position 370 was located in the known effective epitopic region of the NP. In conclusion, although NDV evolved at a high rate and showed great genetic diversity, the structure and function of the NP had been well conserved. However, R0>1 suggests that NDV might have been causing an epidemic since the time of radiation.

Seroprevalence of brucellosis in sheep in the Aksu Region of Xinjiang Uygur Autonomous Region, People's Republic of China, between 1990 and 2010

Seroprevalence of brucellosis infection in sheep between 1990 and 2010 in the Aksu Region, Xinjiang Uygur Autonomous Region, People’s Republic of China was determined by Rose Bengal Precipitation Test of Brucellosis (RBPT). The sera samples were analyzed according to region, husbandry practice, sex, and age. A total of 208,438 sheep were tested, with overall brucellosis seroprevalence of 0.26% (550/208438). No statistically significant difference was found in terms of age and region. Seroprevalence in male sheep (0.53%, 52/9877) was higher than that in the females (p = 0.02; OR = 2.105, 95%; CI = 1.581–2.803). Significant difference (p = 0.03; OR = 2.358, 95%; CI = 1.843–3.017) was evident for rates for brucellosis in the scale breeding (0.13%, 73/55195) and backyard groups (0.31%, 477/153243). The current brucellosis serosurvey is expected to broaden the understanding of zoonotic infection in the region. We concluded that the control measures for brucellosis in the Aksu region sheep herds are effective.