Proteolytic enzyme activity and attenuation of virulence in Theileria annulata schizont-infected cells

A field isolate of Theileria annulata (Uzbek strain) was obtained from calves infected by Hyalomma anatolicum ticks collected from an endemic region in Uzbekistan. Schizont-infected bovine cells that had been established and propagated in cell culture were examined for attenuation both in vivo, by inoculating cells from various passages into calves, and in vitro for metalloproteinase activity. During serial subcultivation a gradual reduction in virulence and in enzyme activity in cells infected with the Uzbek strain were observed. Complete attenuation of the Uzbek isolate was obtained at about passage 80, and only traces of proteolysis were detected in gelatin substrate gels. In contrast, there was no direct correlation between virulence and enzyme levels in an Israeli strain. While schizonts of the Israeli strain were completely attenuated at passage 80, proteolysis in the substrate gels was detected up to passage 197. Solid immunity was observed in calves immunized with attenuated T. annulata schizonts of the Uzbek strain upon challenge with the homologous H. excavatum sporozoites. For a strain to be used for vaccine production, it appears that animal inoculation still remains the most reliable method to assess the degree of attenuation and protection.     

Studies on the potential of immunoprophylaxis using Theileria annulata attenuated by cobalt-60 irradiation in bovine lymphocytes

The possibility of attenuation of the schizontal stage of Theileria annulata within bovine lymphocytes was investigated using ⁶⁰Co radiation at 0,5 and 10 kR levels. Irradiated parasites inoculated into susceptible cross-bred calves induced a relatively non-severe disease with low parasitaemia, mortality and morbidity. Recovered calves were resistant to subsequent tick challenge with the same virulent strain. The degree of attenuation or damage of schizonts appeared to be proportional to the radiation dose. Immunoprophylaxis of susceptible cattle with γ-irradiated T. annulata schizonts seems to be potentially practical under laboratory conditions.     

Effects of rapid passage in the gerbil (Meriones unguiculatus) on the course of infection of the bovine piroplasm Babesia divergens in splenectomised calves

The antigenicity and virulence of the cattle piroplasm Babesia divergens was compared in splenectomised calves before and after adaptation to the gerbil. It was apparent that adaptation of B divergens to the gerbil did not result in attenuation of the parasite in cattle. The gerbil-adapted parasite had a shorter prepatent period, produced higher parasitaemias, and more severe haematological and biochemical changes than the initial bovine strain. The antigenicity of the parasite was unchanged by gerbil passage. The changes in the virulence of the gerbil-adapted parasite were attributed to selection of a rapidly replicating parasite.     

Escherichia coli heat-stable enterotoxin in feces and intestines of calves with diarrhea

Two experiments were conducted to evaluate detection of Escherichia coli heat-stable enterotoxin (ST) in the feces of calves as a method for implicating E coli in neonatal calf diarrhea. The first experiment evaluated the use of the infant mouse test for detection of ST in the feces of calves with naturally occurring diarrhea. Simultaneous identification of bovine enteropathogenic strains of E coli (EEC) and of other infective agents implicated in neonatal calf diarrhea was attempted in these samples. The ST was detected with certainty in only 7 of 41 samples from calves less than or equal to 3 weeks old. Enteropathogenic E coli, however, was detected in 27 samples. In 23 of these 27 samples, EEC was the only recognizable diarrheagenic agent. In a small percentage of the samples, Salmonella, rotavirus, coronavirus, and cryptosporidium were recognized alone, in combination with each other, or with EEC. In the second experiment, 6 calves were fed colostrum from cows inoculated with the bovine EEC strain B44; 6 were given colostrum from cows vaccinated with non-EEC strain 28F, and 4 were given milk from nonvaccinated heifers. Two of the calves that were given colostrum from cows inoculated with strain B44 were challenge exposed with the non-EEC strain 28F. The remaining calves were challenge exposed with the EEc strain B44. Fecal samples were taken from these calves at intervals and were examined for the presence of ST and of the challenge-exposure organism. The ST was detected in approximately one half of the fecal samples obtained, and it was most often detected in the early stages of the induced diarrhea. Calves were observed to shed the challenge-exposure EEC strain for long periods in the absence of diarrhea or detectable amounts of ST in the feces. The ST was detectable in fecal samples when the diarrhea was severe and when the dry matter content of the fecal samples was low.     

Comparison of the pneumopathogenicity of two strains of bovine viral diarrhea virus

The pneumopathogenicity in calves of 2 strains of bovine viral diarrhea (BVD) virus, isolate 2724 (a noncytopathogenic virus) and isolate 72 (a cytopathogenic virus), was compared. All calves were inoculated endobronchially, using fiberoptic bronchoscopy. Two calves were given Pasteurella haemolytica, 2 calves were given the noncytopathogenic BVD virus, and 2 calves were given cytopathogenic BVD virus. Five calves were inoculated sequentially with BVD virus and, 5 days later, with P haemolytica. Two of these calves were inoculated with the noncytopathogenic BVD virus and the other 3 with the cytopathogenic strain. Both BVD virus strains caused marked respiratory tract disease in the calves sequentially inoculated with P haemolytica and also impaired pulmonary clearance of P haemolytica. However, the effect of the cytopathogenic strain was more severe than the noncytopathogenic strain, indicating that strains of BVD virus may vary in their pneumopathogenicity for calves.     

Airborne transmission of bovine herpesvirus 1 infections in calves under field conditions

A small scale transmission experiment was performed with bovine herpesvirus 1 (BHV1) in a cattle population under field conditions. 10 calves were housed under strict hygienic conditions, with a distance of 4m between each calf. Five calves were experimentally infected with BHV1, two calves with strain Harberink and three with strain Lam, respectively. Experimentally infected calves were placed at 4 m distance from five susceptible sentinel calves. Airborne transmission to sentinel calves was detected using virus isolation and BHV1 specific polymerase chain reactions in samples of nasal fluids, and BHV1 specific antibodies in serum samples. Strain Harberink was hardly transmitted to sentinel calves, whereas strain Lam was transmitted to all sentinels. Estimating the rate of transmission per day, the total number of calves infected by one (strain Lam) infected calf was 1.18. Comparing this estimated transmission ratio between cattle at a distance of 4 m to the estimated transmission ratio R of BHV1 in susceptible commingled cattle reported before, the effect of the factor distance on the transmission ratio could be calculated. Extrapolating these results, a distance of 4.4 m between cattle populations would be necessary to reduce transmission for this strain to R<1.     

猪肠道中产蛋白酶解淀粉芽孢杆菌的筛选、鉴定及其酶学性质的初步研究

试验从健康猪肠道中筛选得到一株高产蛋白酶的芽孢杆菌MY-6菌株,通过形态学观察、生理生化试验和糖醇发酵试验、16S rDNA序列扩增与比对、Biolog鉴定等方法鉴定该菌株为解淀粉芽孢杆菌。并研究了蛋白酶活力与细菌生活周期的动态关系,pH、温度、金属离子和蛋白酶抑制剂对蛋白酶活力的影响及蛋白酶对不同底物的水解性能。结果显示,MY-6菌株蛋白酶的合成与其芽孢形成同步,均开始于对数生长期的前期,该菌株所产蛋白酶的最适作用条件为：40℃、pH 7.0,Ca²⁺和Mn²⁺对蛋白酶活力有明显的促进作用,PMSF能够完全抑制该蛋白酶的活力,说明该蛋白酶属丝氨酸蛋白酶,蛋白酶对鱼粉蛋白、大豆蛋白、玉米蛋白、酪蛋白的酶解物TCA可溶性肽含量分别为0.83、0.67、0.58和0.38 μmol/g。结果提示,解淀粉芽孢杆菌MY-6菌株可作为改善蛋白质消化的益生性芽孢杆菌的初步选择。     

Salmonella dublin experimental infection in calves: protection after oral immunization with an auxotrophic aroA live vaccine

Salmonella dublin strain SL5631, which is auxotrophic for p-amino-benzoic acid and 2,3-dihydroxybenzoate because of a deleted aroA gene, was given orally in a dose of 10(10) live bacteria to 6 calves 5-7 weeks old. The calves tolerated the strain well, had a transient mucoid diarrhea and sacrificed animals showed a moderate acute inflammation in the ileum on day 2. The salmonella strain was seen lining the mucosal epithelium using immunohistopathology. Already in calves sacrificed on day 6 the damage was less pronounced and signs of regeneration were obvious. The healing process was more accentuated in calves sacrificed on day 14. The results demonstrated the attenuating effect of the deleted aroA gene. Groups of 5-7 weeks old calves (n = 25) orally immunized with 10(8), 10(9) and 10(10) S. dublin SL5631 at weekly intervals were challenged 2, 6 or 15 weeks after the immunization. All calves were protected against oral challenge with 10(10) bacteria of the virulent S. dublin strain, which equals 1,000 LD50 doses. At autopsy, calves were sacrificed 3 weeks after challenge, all calves had normal intestinal findings with only slightly enlarged mesenteric lymph nodes. The protective effect is surmised to involve cell-mediated as well as humoral defense mechanisms.     

Screening and Identification of Protease-producing Bacillus amyloliquefaciens from Pig's Intestines and the Preliminary Study of Its Enzymatic Properties

A Bacillus strains,MY-6 strain which secreted high amount of protease was isolated from healthy pig's intestines,and it was identified as Bacillus amyloliquefaciens by the methods of morphological observation, biochemical tests,16S rDNA sequence amplification and comparison, biolog identification. In order to get an initial understanding of the enzymatic properties of its protease, the dynamic relationship between protease activity and bacterial life cycle, effects of pH,temperature,metal ions and inhibitors on protease activity,and the protease hydrolysis of different substrates were investigated. The results showed that,the protease synthesis of MY-6 strain was synchronized with its spore formation which began in the early stage of the exponential growth phase. The optimum conditions for the protease was 40℃ and pH 7.0,Ca²⁺ and Mn²⁺ had a intensive effects on the protease activity, PMSF could completely inhibit the activity of protease, indicating that the protease was a serine protease.The content of TCA soluble peptides for protein hydrolyzate fish protein, soybean protein, corn protein and casein was 0.83,0.67,0.58 and 0.38 μmol/g,respectively. In conclusion,Bacillus amyloliquefaciens MY-6 strain could be used as an initial selection of probiotic Bacillus to improve protein digestion.     

Two new variants of the Rhodococcus equi virulence plasmid, 90 kb type III and type IV, recovered from a foal in Japan

Calves were vaccinated orally, subcutaneously or intraperitoneally with a smooth, plasmid-cured strain of Salmonella enterica serovar typhimurium, strain 81. Oral vaccination was not effective, as only 1/5 calves survived challenge with virulent S. typhimurium. Strain 81 was attenuated for calves, as only a slight rise in rectal temperatures was detected after vaccination. The organism was excreted by some calves in the faeces, but no signs of diarrhoea were observed after vaccination. After parenteral vaccination, strain 81 was able to reach the intestines, gastric associated lymphoid tissues and other internal lymphoid tissues and remained viable for up to 14 days in the bovine host. After oral challenge with a virulent strain, 9/10 vaccinated calves survived challenge as opposed to 4/10 control calves (p<0.5). Diarrhoea was present in all calves of the control groups, but in only 4/10 of the vaccinated calves. The clinical reactions of the vaccinated calves were milder than in the control calves, as the rises in rectal temperatures were lower, diarrhoea was less severe, and the challenge strain was present in fewer organs from vaccinated calves than control calves. This study showed that parenterally administered Salmonella vaccines can induce both mucosal and systemic immunity, and it is postulated that this capability of strain 81 is related to its colonisation of lymphoid tissues and other systemic and intestinal tissues. This study confirmed that plasmid-cured strains were attenuated in the bovine host and conferred significant protection after parenteral vaccination, but not oral vaccination.     

Effect of different methods of maintenance on the pathogenicity and infectivity of Babesia bigemina for the vector Boophilus microplus

Observations were made on the effects of five different methods of laboratory maintenance on the infectivity and virulence of Babesia bigemina for the tick Boophilus microplus. The original isolate was highly infective and virulent, causing premature death of engorged female ticks and reduced egg production. Maintenance of the strain by syringe passage in unsplenectomised calves at six to 10 week intervals reduced both its infectivity and virulence for ticks. When slow passages were preceded by a series of rapid passages in splenectomised calves, the changes to the strain were less pronounced. The other three procedures, rapid syringe passage in splenectomised calves and tick passage in either splenectomised or intact calves, had no statistically significant effect on the characteristics measured.     

The vaccination and challenge with bovine viral diarrhea virus (BVDV) of calves previously infected with a non-cytopathic BVDV.

Four calves were infected with noncytopathic (NCP) New York-1 strain of bovine viral diarrhea virus (BVDV). During the observation period of one month the calves remained clinically normal but the virus was repeatedly recovered from their pharyngeal swabbings and blood. Thirty days following infection the four calves were vaccinated, together with two uninfected calves, with a modified-live vaccine containing cytopathic (CP) BVDV, infectious bovine rhinotracheitis virus and parainfluenza-3 virus. No detrimental effects were observed after vaccination. Forty-three days after vaccination the calves were challenged by exposure either with the CP TVM-2 strain or the NCP New York-1 strain of BVDV. The vaccinated calves remained healthy throughout the 60-day observation period.     

牛支原体NM2012株致病性研究

[背景]牛支原体是牛呼吸道疾病综合征的重要病因之一,可以引起牛的多种呼吸道疾病,给养殖业带来巨大的经济损失。接种疫苗是预防牛支原体感染的最有效的手段。[目的]通过人工感染牛体试验,了解牛支原体NM2012株的致病性,为后续疫苗研究提供生产用菌种。[方法]将牛支原体NM2012株第6代、第12代、第20代菌株分别经人工气管注射感染2周龄犊牛,攻毒后连续观察27 d,记录攻毒后犊牛的临床症状,在感染后第14天,从各攻毒组分别选取3头犊牛、空白对照组选取2头犊牛进行扑杀,第27天将剩余实验犊牛处死,观察肉眼病理变化和组织学病理变化。[结果]3个代次的NM2012菌株均对犊牛具有致病性,可引起比较典型的牛支原体肺炎症状和病理变化。[结论]NM2012株6-20代仍然具有对牛的致病性,可以作为今后疫苗研究的潜在菌种。     

Establishment of an attenuated strain of bovine respiratory syncytial virus for live virus vaccine

To develop a live virus vaccine for the prevention of bovine respiratory syncytial (BRS) virus infection in calves, an attempt was made to produce an attenuated virus. The RS-52 strain of BRS virus, isolated from the nasal secretions of a naturally infected calf, was subjected to serial passages in adult hamster lung established (HAL) cells at 30 degrees C and the attenuated rs-52 strain as a live virus vaccine was established. The rs-52 strain multiplied better at 30 degrees C than at 34 or 37 degrees C in HAL cells. The differences in the highest virus titers of this strain between the culture temperature of 30 degrees C and that of 34 or 37 degrees C were more than 2.25 log TCID50. Colostrum-deprived newborn calves and 2 approximately 4 months old calves inoculated with the rs-52 strain manifested no abnormal clinical sings at all. However, all inoculated calves produced serum neutralization antibody. When the colostrum-deprived newborn calves immunized with the rs-52 strain were challenged with the virulent NMK7 strain of BRS virus, they exhibited no pyrexia or other abnormal clinical signs at all. An attempt was made to recover the virus from nasal secretions of these calves, but in vain. On the other hand, a nonimmunized control colostrum-deprived newborn calf developed slight fever, mild cough, and slight serous nasal discharge after challenge exposure. The virus was recovered from nasal secretions of this calf. From these results, it was considered that the rs-52 strain could be used as an attenuated live virus vaccine for prevention of BRS virus infection.     

THE SUSCEPTIBILITY OF YOUNG AND NEWBORN CALVES TO BOVINE EPHEMERAL FEVER VIRUS

The pathogenicity of a field strain, 417, of bovine ephemeral fever (BEF) virus for newborn and young calves was investigated. Three colostrum-deprived newborn calves inoculated intravenously developed severe clinical disease and viraemia, and produced long-lasting neutralising antibody. The incubation period in these animals was 10 and 11 days, compared with 5 to 7 days for older calves. Two newborn calves which received colostrum from immune dams and 2 which received colostrum from non-immune dams failed to respond clinically to intravenous inoculation with strain 417. The neutralising antibody response of these calves was of short duration. Four calves, 7 to 8 weeks old and lacking detectable neutralising antibody to BEF virus, or having low levels of antibody, did not develop clinical disease when inoculated intravenously. Four calves 12 to 14 weeks of age and free of detectable neutralising antibody to BEF virus developed clinical disease when inoculated with strain 417.     

Auxotrophic, plasmid-cured Salmonella enterica serovar typhimurium for use as a live vaccine in calves

Calves were vaccinated orally, subcutaneously or intraperitoneally with a smooth, plasmid-cured strain of Salmonella enterica serovar typhimurium, strain 81. Oral vaccination was not effective, as only 1/5 calves survived challenge with virulent S. typhimurium. Strain 81 was attenuated for calves, as only a slight rise in rectal temperatures was detected after vaccination. The organism was excreted by some calves in the faeces, but no signs of diarrhoea were observed after vaccination. After parenteral vaccination, strain 81 was able to reach the intestines, gastric associated lymphoid tissues and other internal lymphoid tissues and remained viable for up to 14 days in the bovine host. After oral challenge with a virulent strain, 9/10 vaccinated calves survived challenge as opposed to 4/10 control calves (p<0.5). Diarrhoea was present in all calves of the control groups, but in only 4/10 of the vaccinated calves. The clinical reactions of the vaccinated calves were milder than in the control calves, as the rises in rectal temperatures were lower, diarrhoea was less severe, and the challenge strain was present in fewer organs from vaccinated calves than control calves. This study showed that parenterally administered Salmonella vaccines can induce both mucosal and systemic immunity, and it is postulated that this capability of strain 81 is related to its colonisation of lymphoid tissues and other systemic and intestinal tissues. This study confirmed that plasmid-cured strains were attenuated in the bovine host and conferred significant protection after parenteral vaccination, but not oral vaccination.     

THE SUSCEPTIBILITY OF CALVES TO INFECTION WITH A STRAIN OF BOVINE EPHEMERAL FEVER VIRUS INOCULATED INTRACEREBRALLY

Three newborn calves were inoculated intracerebrally with bovine ephemeral fever virus strain 525. The 2 that lacked detectable neutralising antibody to bovine ephemeral fever vaccine developed fatal encephalitis after 4 and 7 days respectively. The third calf which had a low level of maternal antibody remained healthy and developed antibody that became undetectable after 6 months. Bovine ephemeral fever virus strain 525 was reisolated from the brains of both dead calves by intracerebral inoculation of suckling mice. Homogenates that were prepared from the brains of the calves failed to produce disease or to induce antibody formation in susceptible calves when inoculated intravenously. Strain 525 of BEF virus has been shown to possess a degree of neurovirulence for laboratory animals that has not been reported for other strains (Tzipori and Spradbrow 1974). Although this strain is unable to produce viraemia in calves after I/V inoculation, the present study shows that strain 525 can multiply in the brain tissues of calves and cause death after I/C inoculation.     

Intestinal and extra-intestinal pathogenicity of a bovine reassortant rotavirus in calves and piglets

Despite the impact of bovine group A rotaviruses (GARVs) as economically important and zoonotic pathogens, there is a scarcity of data on cross-species pathogenicity and extra-intestinal spread of bovine reassortant GARVs. During the course of characterizing the genotypes of all 11 genomic segments of bovine GARVs isolated from diarrheic calves in South Korea, a unique G6P[7] reassortant GARV strain (KJ9-1) was isolated. The strain harbors five bovine-like gene segments (VP7: G6; VP6: I2; VP1: R2; VP3: M2; NSP2: N2, and NSP4: E2), five porcine-like gene segments (VP4: P[7]; NSP1: A1; NSP3: T1, and NSP5: H1), and one human-like gene segment (VP2: C2). To investigate if this reassortant strain possessed cross-species pathogenicity in calves and piglets, and could induce viremia and extra-intestinal spread in calves, colostrum-deprived calves and piglets were experimentally inoculated with the KJ9-1 strain. The KJ9-1 strain caused severe diarrhea in experimentally infected calves with extensive intestinal villous atrophy, but replicated without causing clinical symptoms in experimentally infected piglets. By SYBR Green real-time RT-PCR, viral RNA was detected in sera of the calves at post-inoculation day (PID) 1, reaching a peak at PID3, and then rapidly decreasing from PID4. In addition, viral RNA was detected in the mesenteric lymph node, lungs, liver, choroid plexus, and cerebrospinal fluid. An immunofluorescence assay confirmed viral replication in the extra-intestinal organs and tissues of virus-inoculated calves. The data indicates that the homologous/heterologous origin of the NSP4 gene segment (E2 genotype), may play a key role in the ability to cause diarrhea in calves and piglets.     

An experimental contribution to the study of the pathogenesis of bovine viral diarrhea virus infection.

This presentation summarizes the results of a study on the pathogenesis of bovine viral diarrhea (BVDV) infection. The cytopathic (CP) strain TVM-2 of BVDV induced in calves an overt clinical disease which is usually recorded as the acute primary BVDV infection observed under natural conditions. In contrast the non-cytopathic (NCP) strain New York-1 of BVDV did not cause any significant signs of disease. However, when the calves were immunosuppressed by treatment with dexamethasone (DMS) the biotype of BVDV involved did not seem to be as important as it appeared to be in an immunologically normal animal. This was shown in this study by the NCP BVDV which caused a fatal disease in calves treated with DMS. A mixed infection given to calves by injecting them with both CP and NCP BVDV, did not result in any particularly serious disease. So, the potential immunosuppressive activity of BVDV itself for the host has not been proven under the experimental procedures used in this experiment. Finally, a modified-live CP BVDV vaccine was unable to cause clinical disease when injected into calves that had been infected previously with strain New York-1 of BVDV.     

Attenuation of a virulent type 2 bovine viral diarrhea virus

The purpose of this study was to produce an attenuated bovine viral diarrhea virus (BVDV) type 2 strain as a tool for identifying potential virulence markers in the BVDV2 genome. The attenuation of the virulent strain, BVDV2-24515, was accomplished by in vivo and in vitro passage. The strain was initially used to infect an elk (Cervus elaphus) [J. Wildl. Dis. 35 (1999) 671], re-isolated at 7 days post-inoculation from serum, and then subsequently passaged 56 times in cell culture. Two groups of calves were inoculated intranasally with either BVDV2-24515 or the putative attenuated virus, designated BVDV2-LATT. Calves inoculated with BVDV2-24515 had cumulative clinical scores which ranged from 6 to 53. Clinical signs in these calves consisted of anorexia, depression, dehydration, diarrhea (±bloody), and pneumonia. Several calves developed leukocytopenia, primarily a neutrocytopenia, and presented lesions of enteritis or pneumonia at necropsy. In contrast, cattle inoculated with BVDV2-LATT had cumulative clinical scores which ranged from 0 to 2. This was not significantly different from that of controls which received no virus (range: 0–1). Calves inoculated with BVDV2-LATT produced high neutralizing antibody titers against BVDV2. Thus, in addition to its potential use as a tool for identifying virulence markers, the attenuated virus is also worthy of further study as a candidate virus for inclusion in a modified-live vaccine.