Effect of modified oligopeptides from the beetle Allomyrina dichotoma on Escherichia coli infection in mice

The novel peptides based on a putative active site of defensin, an anti-bacterial peptide from the beetle Allomyrina dichotoma, were synthesized. These synthetic oligopeptides exhibited strong anti-bacterial activity in vitro, even against antibiotic-resistant pathogenic bacteria. Then, anti-bacterial activity of two newly synthesized peptides, RLYLRIGRR-NH(2) (peptide A) and RLRLRIGRR-NH(2) (peptide B) was also examined by macroscopic and histopathologic assessment during the course of infection in mice inoculated with antibiotic-resistant pathogenic Escherichia coli (E. coli) in vivo. Peptide B decreased the mortality of mice inoculated with antibiotic-resistant pathogenic E. coli. The results of macroscopic and histopathologic examinations revealed that peptide B could protect the mice from infection. In contrast, peptide A failed to protect mice from infection with antibiotic-resistant pathogenic E. coli. Also, modified peptides A and B produced no toxicity or side effects in mice. These results suggest that peptide B is useful for developing novel antibiotics against antibiotic-resistant pathogenic bacteria.     

Changes of splenic lymphocyte subpopulation in mice inoculated with Babesia microti and Babesia rodhaini.

Changes of splenic lymphocyte subpopulation after Babesia microti and Babesia rodhaini inoculation in mice were examined by flow cytometric analysis. The B. microti inoculated mice showed a longer period of time from inoculation to the onset of increase or decrease parasitaemia (%), packed cell volume, total spleen cell numbers and surface immunoglobulin positive splenic cell numbers than respective periods in B. rodhaini inoculated mice. The Thy-1 positive cell numbers in B. microti inoculated mice and B. rodhaini inoculated mice pre-immunized with homologous parasites were significantly higher than that of B. rodhaini inoculated mice. The ratio of L3T4 positive cell/Lyt-2 positive cell after inoculation with B. microti was quite similar to that in B. rodhaini mice pre-immunized. However, the ratio in B. rodhaini inoculated mice revealed a lack of an increasing phase. These results suggested that the T-cell dependent early immune response, especially suppressor activity, was closely related to the difference in the course of infection between the non-lethal B. microti and the lethal B. rodhaini infection in mice.     

Antimicrobial activity of a bovine myeloid antimicrobial peptide (BMAP‐28) against methicillin‐susceptible and methicillin‐resistant Staphylococcus aureus

A bovine myeloid antimicrobial peptide (BMAP‐28) is a member of the cathelicidin family which is included in the innate immune system of mammals. Recently, there have been many studies about antimicrobial peptides. This study aims to clarify whether BMAP‐28 has bactericidal activity against methicillin‐resistant Staphylococcus aureus (MRSA) and compares its activity against methicillin‐susceptible S. aureus (MSSA) and MRSA. We found that the peptide was effective in killing MRSA (minimal inhibitory concentration (MIC) range; 5–20 µg/mL). It was also revealed that MSSA (MIC range; 1.25–20 µg/mL) had two levels of susceptibility to BMAP‐28. We also examined the effect of BMAP‐28 on bacterial shape to visually show its activity. After exposure to the peptide, both MSSA and MRSA cells showed the morphological changes on their surfaces. Our results indicate that BMAP‐28 is a promising candidate for medicine against drug‐resistant bacteria.     

Viral replication and lesions in BALB/c mice experimentally inoculated with porcine circovirus isolated from a pig with postweaning multisystemic wasting disease

Eight-week-old BALB/c mice were either sham inoculated (control mice) or were inoculated intraperitoneally (IP) and intranasally (IN) with a single (sPCV mice) or multiple (mPCV mice) doses of porcine circovirus 2 (PCV2). Four control mice and 4 sPCV mice were sacrificed 7, 14, 28, and 42 days postinoculation (PI). All 4 mPCV mice were sacrificed 42 days PI. In addition, 7-day and 14-day pregnant BALB/c mice were either sham inoculated (control mice) or were inoculated IP and IN with a single dose of PCV2. Newborn mice were euthanatized 1, 8, and 15 days after birth. Necropsies were performed on all euthanatized mice and tissues were collected for histopathology, electron microscopy, in situ hybridization, and polymerase chain reaction (PCR). PCV2 replicated in 8-week-old BALB/c mice that were inoculated with PCV2 and caused fetal infection when inoculated into pregnant BALB/c mice at 7 days and 14 days of gestation. PCV was detected by in situ hybridization and PCR in sPCV mice on days 7, 14, 28, and 42 PI; in mPCV mice on day 42 PI; and in newborn mice from mothers inoculated with PCV at 7 days and 14 days of gestation at 1, 8, and 15 days after birth, but not in control mice. No clinical signs or gross lesions were found in sPCV or mPCV mice during the study. Microscopic lesions in sPCV mice and mPCV mice were characterized by expansion of germinal centers in lymphoid organs with large numbers of histiocytic cells and lymphoblasts, apoptosis of histiocytic cells in germinal centers, and mild lymphoid depletion of the paracortex. PCV nucleic acid was detected in the nuclei and cytoplasm of histiocytes and apoptotic cells in germinal centers in lymphoid tissues as well as in the nuclei of hepatocytes in the liver, in the nuclei of renal tubular epithelial cells, and in the cytoplasm of single lymphocytes in the thymus. Congenitally infected mice only had PCV nucleic acid detected in putative Kupffer cells in livers.     

Effect of Eimeria tenella infection on the caecal population of lincomycin-resistant Clostridium perfringens introduced into chickens

The effect of Eimeria tenella infection on the caecal population of lincomycin-resistant Clostridium perfringens (KGW 851) newly introduced into chickens was studied. Four groups of chickens consisted of: (1) Uninoculated controls; (2) inoculated with C perfringens (KGW 851); (3) inoculated with E tenella; and (4) inoculated with C perfringens (KGW 851) followed by E tenella. Five chickens in each group were necropsied on each of days 0, 3, 5, 7, 10 and 14 after the E tenella inoculation. The mean total C perfringens counts in the caecal contents increased at five days after E tenella inoculation and reached maximum counts at seven days after the inoculation. Also lincomycin-resistant C perfringens readily established itself at approximately one 10th of the total C perfringens population in the presence or absence of E tenella infection.     

Efficacy of avian pneumovirus vaccines against an avian pneumovirus/Escherichia coli O2:K1 dual infection in turkeys

The clinical, pathological and microbiological outcome of a challenge with avian pneumovirus (APV) and Escherichia coli O2:K1 was evaluated in turkeys vaccinated with an attenuated APV vaccine and with or without maternally derived antibodies. Two groups of two-week-old poults, one with and one without maternally derived antibodies against APV, were vaccinated oculonasally with attenuated APV subtype A or B. A third group remained unvaccinated. Eleven weeks later, the turkeys were inoculated intranasally with either virulent APV subtype A, or E. coli O2:K1, or with both agents three days apart. After the dual infection, birds vaccinated with attenuated subtype A or B, and with or without maternally derived antibodies, had lower mean clinical scores than the unvaccinated birds. In the vaccinated birds, virus replication was significantly reduced and no bacteria were isolated, except from the birds vaccinated with attenuated subtype B. In the unvaccinated turkeys, large numbers of E. coli O2:K1 were isolated from the turbinates of the dually infected birds between one-and-a-half and seven days after they were inoculated.     

Effects of T-2 mycotoxin on tumor susceptibility in mice

The effect of Fusarium-produced T-2 toxin on tumor growth was evaluated in ICR, CFW, and C57B6/6 mice inoculated with murine sarcoma, Ehrlich ascites carcinoma, or B16F1 melanoma tumor cell lines. Mice were given T-2 toxin intragastrically either at the rate of 2 mg of toxin/kg of body weight daily for 5 days or a single dosage of 4 mg of toxin/kg and were inoculated SC with tumor cells 1 or 2 days after administration of toxin. Tumor growth was assessed 15 to 41 days after tumor challenge by determining the frequency of tumor development and tumor weights. Significant increases in the frequency of development of murine sarcoma (P less than 0.005), Ehrlich ascites carcinoma (P less than 0.01), and B16F1 melanoma tumors (P less than 0.05) were detected in toxin-treated mice, compared with control mice. Murine sarcoma and B16F1 melanoma tumor weights also were significantly (P less than 0.01) higher in toxin-treated mice. The effect of T-2 toxin on tumor growth was more marked after 5 daily treatments than after a single dose.     

Immune responses of immunocompetent and immunocompromised mice experimentally infected with Bartonella henselae

The aim of this study is to understand host immune responses in immunocompetent and immunocompromised mice against Bartonella henselae infection. BALB/c and nude (BALB/c nu/nu) mice were inoculated intraperitoneally with 10(8) colony forming units of B. henselae (Houston-1 strain). Blood, brain, liver, spleen, kidney and bone marrow samples were collected 0, 3, 7, 14, 21 and 28 days after infection and submitted to bacteriological, serological and genetical examinations. B. henselae was isolated only from the liver 3 days after infection. DNA of the inoculums was detected by polymerase chain reaction from blood, liver, and spleen samples collected from BALB/c and blood from nude mice 3 and 7 days after infection. No bacterial DNA was detected from both BALB/c and nude mice thereafter during 4 weeks observation periods. These results indicate that the T-cell may not participate in the effective elimination of the organisms from mice. In addition, western blot analysis revealed that the antigens of 27.3- and 31.5-kDa reacted with IgM antibodies from the blood of BALB/c and nude mice after 3 days of infection, suggesting that these antigens were recognized by thymus-independent mechanism. Furthermore the antigens were detected from the culture-supernatants of B. henselae, indicating that these antigens were secreted from the organisms.     

Effect of salinity, temperature and storage time on mouse experimental infection by Cryptosporidium parvum

Cryptosporidium parvum oocysts collected from a naturally infected calf were exposed to different salinity and temperature for 2, 21 and 40 days, and then inoculated intragastrically into coccidium-free neonatal mice. The intensity of infection as determined seven days later by examination of intestinal homogenates were statistically analysed. Salinity, time and salinity-time interaction were the only factors with significant effect on the infection intensity.     

Experimentally induced infection of cats with Borrelia burgdorferi.

To determine whether cats could be infected experimentally with Borrelia burgdorferi, 15 cats were inoculated with approximately 1,000 B burgdorferi. Seven cats were inoculated by the IV route, 2 by the oral route, 2 by the ocular route, and 4 by the oral-ocular route. Six control cats were inoculated with phosphate-buffered saline solution by the IV, oral, and ocular routes. Prior to the start of the study, all 21 cats were seronegative for B burgdorferi on the basis of results of the indirect fluorescent antibody (IFA) test, and their blood was B burgdorferi culture negative. All of the IV, orally, and ocularly inoculated cats developed IgG antibodies to B burgdorferi as detected by IFA testing. Of 4 oral-ocularly inoculated cats, 2 developed IFA-detectable antibodies and the remaining 2 cats developed low-titer response (1:128) on postinoculation (PI) day 10 only. All control cats remained seronegative. The organism was detected in blood smears from 2 of the IV inoculated cats on PI days 10 and 24 and from 2 oral-ocularly infected cats, 1 on PI days 17 and 24 and 1 on PI day 10. Spirochetes were not detected in the blood after PI day 24. The organism was isolated from tissues of only 1 cat (the lung of an ocularly inoculated cat necropsied at 7 months after inoculation). Spirochetes were not isolated from control cats. Neither clinical signs of infection nor gross or histologic abnormalities were found in any of the inoculated or control cats. Results indicate that cats are susceptible to infection with B burgdorferi, but clinically apparent disease may not be common.     

The primary GP5 neutralization epitope of North American isolates of porcine reproductive and respiratory syndrome virus

I have used indirect ELISA with overlapping synthetic peptides representing the GP5 ectodomain to study the generation and specificity of peptide-binding Abs in pigs that were infected in utero with porcine reproductive and respiratory syndrome virus (PRRSV) strain VR2332 and in North American field sera submitted for PRRSV infection diagnosis. Peptide-binding Abs appeared in sera of the VR2332-infected pigs within about 30 days post-farrowing (dpf), reaching maximum titers 100-200 dpf and then decreasing slowly to about half of maximum titer by about 400 dpf. The formation of peptide-binding Abs and of virus neutralizing Abs correlated and their initial appearance coincided with disappearance of virus from the circulation. The Abs were specific for VR2332-specific peptides. In contrast, anti-N-protein Abs as measured by HerdCheck ELISA appeared within 7 dpf, reached maximum levels at about 100 dpf and had decreased below detectable levels by about 200 dpf. Twenty-seven field serum samples with virus neutralizing activity all possessed high levels of peptide binding Abs, but the Abs bound about equally to VR2332 and strain Lelystad virus (LV)-specific peptides. The indirect ELISA results using various large peptides and competition ELISA using small peptides (8 or 9 amino acids long) confirmed that the epitope recognized by the Abs is located in the GP5 ectodomain sequence 37SHLQLIYNL of VR2332. Use of mutated peptides in the competition ELISA showed that 42I to T and 38HL to TY substitutions blocked Ab recognition, whereas deletion of 41L had no effect. In addition, 26 serum samples submitted by two farms for diagnostic tests were found to possess low levels of Abs that bound to GP5 ectodomain peptides, even though the sera were sero-negative in the HerdChek ELISA and lacked neutralizing activity. Competition ELISA showed that the Abs recognized one or more epitopes located downstream of the PRRSV neutralization epitope. An epitope(s) located in the same area was recognized by Abs generated in mice by immunization with a GP5 ectodomain peptide conjugated to BSA. These Abs also lacked neutralizing activity.     

Alymphoplasia mice are resistant to prion infection via oral route

The major cause of infection in animal prion diseases is thought to be consumption of prion-contaminated stuff. There is evidence that the enteric nerve system (ENS) and gut-associated lymphoid tissues (GATL) are involved in the establishment of prion infection through alimentary tract. To elucidate the initial entry port for prion, we inoculated prion to alymphoplasia (aly) mice showing a deficiency in systemic lymph nodes and Peyer's patches. The aly/aly mice were susceptible to prion infection by intra-cranial inoculation and there were no differences in incubation periods between aly/aly mice and wild-type C57BL/6J mice. Incubation periods in aly/aly mice were about 20 days longer than those in C57BL/6J mice with the intra-peritoneal inoculation. The aly/aly mice were completely resistant to prion infection by per os administration, while C57BL/6J mice were sensitive as they entered the terminal stage of disease around 300 days post inoculation. PrP(Sc) were detected in the intestine and spleen of C57BL/6J mice inoculated with prion intraperitoneally or orally; however PrP(Sc) was not detected in the spleen and intestine of aly/aly mice. Prion infectivity was detected in the intestines and spleens of prion-inoculated C57BL/6J mice, even after the early stages of exposure, while no infectivity was detected in these tissues of prion-inoculated aly/aly mice. No apparent differences were observed in the organization of the enteric nerve system between wild-type and aly/aly mice. These results indicate that GALT rather than ENS acts as the primary entry port for prion after oral exposure.     

Desferoxamine and iron dextran in acute Salmonella cholerae-suis infection in pigs

Serum iron (SI)-related and hematologic changes were evaluated in a herd of weaned pigs inoculated with a strain of Salmonella cholerae-suis, causing 83% mortality within 22 days after inoculation was done. Serum iron concentrations decreased to 35% of base-line values 2 days after inoculation was done, but recovered to near base line subsequently. Total SI-binding capacity (TIBC) decreased gradually for 14 days after inoculation was done. Transferrin (TF) concentrations decreased to near half the base line throughout the postinoculation observation period. The calculated SI saturation coefficient decreased to half the base line, but recovered to or above the base-line value subsequently. Combined observations of SI, TIBC, TF, and SI saturation coefficient concentrations indicated that there was higher saturation of host iron-binding proteins and recruitment of additional iron-binding systems subsequent to 2 days after inoculation was done. Day 2 after inoculation seemed to be a critical period for host iron metabolism. Injection of supplemental iron dextran simultaneously with Salmonella infection resulted in lower mortality of iron-injected pigs (P less than 0.005). A highly significant negative correlation was observed between SI concentration and rectal temperatures after pigs were inoculated with Salmonella (r = -0.54; P less than 0.0001). Hemoglobin concentrations, mean corpuscular volume, and mean corpuscular hemoglobin were not significantly affected by Salmonella infection or iron injection concurrent with Salmonella infection.(ABSTRACT TRUNCATED AT 250 WORDS)     

Persistent plaque formation in experimental murine brucellosis

Primary plaque forming cells (PFC) are present in spleens of mice 150 days or more following an infection with Brucella abortus. The development of primary plaques in mice long after antigenic challenge is an uncommon phenomenon, unlike the plaque formation (PF) induced by a non-living antigen. The mechanism of this persistent PF has been now investigated in light of a prolonged persistence of the corresponding antigen in tissues. Living E. coli, inoculated in massive dose into mice, survived in their organs for a brief time, while concomitantly PFC disappeared by day sixteen. Infection with B. abortus, in contrast, induced persistent presence of bacteria in the organs of inoculated mice and stimulated long lasting plaque formation. Only direct plaques were found during all stages of infection. Repeated inoculations of dead B. abortus also induced continuous production of primary plaques, whereas an interval in supply of the antigen resulted in disappearance of PFC. Rifampin (40 mg/kg) eliminated bacteria from the treated mice, which resulted in the disappearance of primary PFC. It seems likely that long lasting PF in B. abortus infected mice is connected with a constant antigenic stimulus operating in the carrier state.     

坏死梭杆菌FN(A)p2001株小鼠感染模型的建立

将鹿源坏死梭杆菌FN(A)p2001分离株以3×108、3×107、3×106、3×105、3×104、3×103个/只等不同菌量,分别接种于6组小鼠,每组10只,逐日观察小鼠感染情况.结果表明,3×106、3×107、3×108个/只菌量感染组小鼠,在接种后3 d～8 d先后死亡,5 d后死亡小鼠的病理变化明显,并从死亡小鼠内脏及脓汁中均检到长丝状及小杆状等多形态典型的坏死梭杆菌;3×104个/只和3×105个/只菌量感染仅表现体重减轻,3×103个/只菌量感染无明显临床表现.由此表明,坏死梭杆菌FN(A)P2001分离株具有很强的感染毒力;对小鼠的最小致死量为106个/只菌量;腹腔接种是适宜的感染途径.从而建立了坏死梭杆菌分离株小鼠感染模型.     

单核细胞增生性李斯特菌感染ICR小鼠模型的建立

单核细胞增生性李斯特菌(Listeria monocytogenes,LM)是一种人畜共患食源性致病菌,能引起人和动物较为严重的感染症状。现广泛使用具有免疫活性的小鼠模型测定半数致死量(LD₅₀)来评价不同LM菌株的致病性。为建立LM的ICR小鼠模型,本研究采用1/2a血清型的N21 LM菌株,分别以10⁶、10⁷、10⁸、10⁹和10¹⁰ CFU的剂量口腔灌注感染6周龄ICR小鼠,每组10只;另取10只接种PBS作为对照组,测定LM对ICR小鼠的LD₅₀;另取40只小鼠,平均分为2组,公母各半,以测定的LD₅₀剂量接种LM,分别对各组小鼠临床症状、组织病理变化、体重变化及组织中细菌载量进行评价。结果发现,N21 LM菌株对ICR小鼠的LD₅₀为10^9.25 CFU;感染周期为10 d左右,感染小鼠出现被毛粗糙、精神萎靡、阴茎垂出及体重下降等临床症状。组织病理学分析结果显示,肝脏先后出现实质内灶状细菌团块、形成血栓、细胞坏死等;脾脏主要表现为白髓内淋巴细胞减少;肺脏主要表现为纤维素性肺炎。菌落计数和Real-time PCR的检测结果发现肝脏中细菌载量最高,脾脏次之。结果表明,ICR小鼠能作为单核细胞增生性李斯特菌的感染模型。本试验结果为研究LM的致病机制、疫苗研究及抗菌肽转基因小鼠抗LM的评价奠定了基础。     

The effect of experimental infection with Corynebacterium pseudotuberculosis on reproduction in adult ewes

Fifteen ewes were inoculated subcutaneously with 2 x 10(6) cells of Corynebacterium pseudotuberculosis seven days before mating, during the embryonic stage of gestation and during the fetal stage of gestation. The clinical signs, antibody response and the consequences of the infection on reproduction were studied. None of the ewes showed any change in general condition during pregnancy. The effects of experimental infection during gestation were evident after incubation periods that ranged from 25 to 140 days after inoculation and were dependent on the time of the inoculation in relation to the period of gestation. Ewes inoculated before gestation can resolve infection. Ewes inoculated during the embryonic stage were severely affected and some aborted. In others lambs were stillborn or, if born alive, infected. Ewes inoculated during the fetal stage of gestation did not show reproductive disorders although some remained chronically infected.