Evaluation of the prophylactic use of ovotransferrin against chlamydiosis in SPF turkeys

Chlamydophila (C.) psittaci infections are highly prevalent in turkeys and the economical and public health importance of these infections has been recognized since 1950. As there are no vaccines, antibiotic treatment (tetracylines, enrofloxacine) is often needed to allow marketing of poultry. In this study, we explored the use of ovotransferrin (ovoTF), a natural anti-microbial protein, in preventing an experimental C. psittaci infection in specific pathogen free (SPF) turkeys. Turkeys were treated with aerosolized ovoTF prior to the infection. Groups 1 and 2 received a single dose of 10 and 5 mg ovoTF per turkey, respectively. Group 3 received a daily dose of 5mg ovoTF per turkey during 12 days. Group 4 served as untreated, infected control group. Turkeys were aerosol infected using 10(6) TCID(50) of the virulent C. psittaci serovar/genotype D strain 92/1293. Birds were monitored (clinical signs, bacterial excretion) during 12 subsequent days before being necropsied. At necropsy, pathology and C. psittaci replication in various tissues was examined. A single dose of 10mg ovoTF and a repeated daily dose of 5mg ovoTF could not prevent the birds from becoming infected with C. psittaci, but they significantly reduced the outcome of the infection. A single dose of 5mg ovoTF had no influence on the outcome of the infection as compared to the non-treated infected controls. Our results demonstrate the anti-chlamydial effect of ovoTF in vivo and present a base for further research on practical applications of ovoTF on turkey farms.     

Trans-endothelial and trans-epithelial transfer of lactoferrin into the brain through BBB and BCSFB in adult rats

The transportation of intravenously administered bovine lactoferrin (bLF) into the cerebrospinal fluid (CSF) was immunohistochemically investigated in adult rats. Administered bLF was detected in the vesicular membranes of endothelial cells in cerebral blood vessels 10 min after the infusion. Numerous immunoreactive small vesicles were also detected at the ependymal cells in the choroid plexus. Moreover, the bLF concentration in the CSF was significantly increased at 1-2 hr after the intravenous infusion of bLF (10 or 30 mg/kg). These findings clearly demonstrate that LF is possibly transported into the brain matter even in adult animals.     

Use of ovotransferrin as an antimicrobial in turkeys naturally infected with Chlamydia psittaci, avian metapneumovirus and Ornithobacterium rhinotracheale

Respiratory pathogens are difficult to control in large-scale turkey production. This report describes a clinical trial of antimicrobial ovoTF aerosol on a large Belgian turkey farm. ovoTF was administered to reduce Chlamydia psittaci (C. psittaci) infections and to study the impact of this action on the occurrence of Ornithobacterium rhinotracheale (O. rhinotracheale) and avian metapneumovirus (aMPV) infections. Two subsequent broods were included; (i) a control brood receiving no ovoTF and (ii) an ovoTF brood receiving ovoTF aerosol (5mg/animal) at the age of 2 weeks, continuing daily for 12 days. Twenty-four one-day-old toms of the control and ovoTF brood were tagged and monitored for 15 weeks. The control brood experienced two periods of respiratory disease, the first (2-3 weeks of age) due to C. psittaci and the second (8-17 weeks of age) in the presence of C. psittaci, O. rhinotracheale and maybe aMPV. Extensive antibiotic treatment was needed in 2, 8 and 9 week-old toms. In the ovoTF brood, toms stayed healthy until the age of 9 weeks, whereafter respiratory disease occurred in the presence of C. psittaci, O rhinotracheale and aMPV. OvoTF administration: (i) reduced the amount of C. psittaci in the air as demonstrated by bioaerosol monitoring, (ii) prevented respiratory disease during the first half of the brood period, (iii) was associated with 46% reduction of mortality, and (iv) reduced the antibiotic cost. Our results justify additional clinical trials to explore the use of this innovative antimicrobial strategy for poultry.     

Effect of bovine lactoferrin on a transmissible AIDS-like disease in mice

The effect of bovine lactoferrin (bLF) was examined on an AIDS-like disease (ALD) in mice. Induction of disease was achieved by inoculation with infected cell-free plasma from diseased mice to uninfected ones. The effect of treatment with bLF was investigated when administered simultaneously with the virus, 20 days prior to infection, or 20 days after infection. Animals underwent clinical surveillance and enumeration of white blood cells (WBC) and lymphocytes, as well as fluorescent staining of CD4 and CD8 bearing cells. Simultaneous administration of bLF and virus did not affect the pattern of ALD progress along the course of the experiment. Pretreatment with bLF prior to virus inoculation abolished on day 21 the detrimental effect of viral infection that lasted for two months. An opposite outcome was observed when bLF was administered 20 days after the virus. It seems that bLF had played a preventive role for a restricted period of time. However, an adverse response was elicited when bLF was administered 20 days after viral infection.     

Dynamics of the development of Chlamydophila psittaci inclusions in epithelial and fibroblast host cells

The development of Chlamydophila psittaci (formerly Chlamydia psittaci, avian strains) inclusions in fibroblast L-929 and epithelial BGM cell lines was studied along the bacterial growth cycle using a BGM cell-adapted strain in the presence or absence of cycloheximide and cycloheximide + polyethylene glycol. Evolution of the inclusions was determined in terms of their number and size at 24, 30, 36, 48 and 54 h after infection. Significant differences in the chlamydial growth were found between both host cells, throughout the study. Higher numbers of inclusions (P < 0.05) were observed in L cells while larger inclusions (P < 0.01) were found in BGM cells. In both fibroblast and epithelial cells, inclusions showed a significant (P < 0.001) increase in size at the later times studied. Free extracellular chlamydial particles were noticed at 48 and 54 h post-infection (p.i.) in infected L cells, and at 54 h p.i. in BGM cells. Addition of cycloheximide or cycloheximide + polyethylene glycol had no significant effect on the number of inclusions or their size. The results suggest that host cell characteristics and innate compatibility between Chlamydophila strain and host cell are more important than host cell adaptation for the development of the microorganism.     

人乳铁蛋白cDNA的克隆及在家蚕细胞中的表达

从人的乳腺组织中克隆了人乳铁蛋白(hLF)cDNA,其DNA序列与GenBank中另外4个hLF cDNA序列具有高度的同源性(同源性达到99%)。将人乳铁蛋白cDNA克隆在质粒pBacPAK8的BamHⅠ位点和XhoⅠ位点,构建成重组转移质粒pBacPAK-hLF。该质粒DNA与已线性化BacPAK6DNA共转染家蚕细胞BmN,在培养的贴壁细胞中挑出空斑,经过3轮纯化,获得重组病毒BmNPV-hLF。蛋白质免疫印迹法检测到在重组病毒感染的家蚕细胞中存在人乳铁蛋白基因的表达产物,分子量约78kD,ELISA法测定结果表明家蚕细胞中重组人乳铁蛋白(rhLF)相对表达量在表达120h达到最高值,约为13.5mg/L。体外生物活性试验表明重组hLF对大肠杆菌JM109具有抑菌活性。     

Development of a Chlamydophila psittaci species-specific and genotype-specific real-time PCR

A Chlamydophila psittaci species-specific real-time PCR targeting the rDNA ribosomal spacer was developed as well as a genotype-specific real-time PCR targeting the Cp. psittaci outer membrane protein A (ompA) gene. The SYBR Green-based species-specific real-time PCR detected Cp. psittaci genotypes A to F, and the recently discovered E/B genotype. The genotype-specific real-time PCR could easily distinguish genotypes C, D, F by use of TaqMan probes. Genotypes A, B and E could not be distinguished from each other by simply using TaqMan probes. For this purpose, non-fluorescent competitor oligonucleotides, had to be used next to the TaqMan probes. Genotype E/B could only be detected by use of a minor groove binder (MGB) probe. Both real-time PCR assays allowed reproducible, sensitive (10 rDNA or ompA copies/microL DNA extract) and specific detection of Cp. psittaci DNA. The genotype-specific real-time PCR was compared to ompA sequencing and ompA restriction fragment length polymorphism (RFLP) analysis using five Cp. psittaci field isolates (99, 61/8, 7344/2, 8615/1 and 7778B15) each consisting of two different genotypes. The currently developed real-time PCR assays were used in a case study on a veterinary school and a turkey farm. In the veterinary school, Cp. psittaci genotypes D, E/B and F infection were detected in all five groups of turkeys, and one veterinarian who was taking care of all these turkeys. On the turkey farm, the presence of two Cp. psittaci genotype B infection waves was demonstrated in one randomly selected turkey, the first wave at the age of 6 weeks, and the second at the age of 12 weeks.     

乳铁蛋白及其水解物对直投式乳酸菌发酵剂生长的影响

以直投式乳酸发酵剂为实验菌,研究乳铁蛋白及其水解产物对发酵剂各生长阶段的影响。实验条件为乳铁蛋白质量浓度范围0.5～2.5mg/mL,乳铁蛋白水解物的质量浓度范围0.05～0.3mg/mL,无氧条件下37℃培养。结果表明,37℃恒温培养,乳铁蛋白在0.5～2.0mg/mL范围内对乳酸菌生长有促进作用,最适添加质量浓度为1.0mg/mL,抑菌质量浓度为大于2.5mg/mL;乳铁蛋白水解物在0.05～0.25mg/mL范围内对乳酸菌生长有促进作用,最适添加质量浓度为0.1mg/mL,抑菌质量浓度为大于0.3mg/mL。     

乳铁素参与乳房链球菌对乳腺上皮细胞的粘附

免疫印迹试验表明，试验用的3株乳房链球菌（Streptococcus uberis)菌株均可与乳铁素（Lactoferrin)结合。培养基中加入乳铁素或乳清可以显著促进细菌与乳腺上皮细胞之间的粘附，抗乳铁素抗体可以特异性地抑制乳铁素或乳清预处理细菌与乳腺上皮细胞的粘附。乳铁素在细菌和细胞之间起着桥梁分子作用。有助于细菌与乳腺上皮细胞之间的粘附和乳腺感染的建立。     

Diagnosis of avian chlamydiosis: specificity of the modified Giménez staining on smears and comparison of the sensitivity of isolation in eggs and three different cell cultures.

For the diagnosis of chlamydiosis in dead and live birds different methods were compared for their sensitivity and specificity. The specificity of the modified Giménez staining and the direct immunofluorescence (DIF) test for direct demonstration of Chlamydia psittaci in organ, cloacal and/or conjunctival smears was examined. The sensitivity of the isolation of Chlamydia psittaci in 6 days embryonated specific pathogen free (SPF) chicken eggs, Buffalo Green Monkey (BGM) cell line, McCoy cell line and Vero cell line was compared. On smears, the direct immunofluorescence test was more specific than the modified Giménez staining. The concordance between the results of both detection methods was 80%. The BGM cell culture was the most sensitive artificial host for isolation of Chlamydia psittaci, followed by the embryonated eggs, the Vero cell line and the McCoy cell line. The concordance between the results of isolation in BGM cell culture and eggs was 96.5%, while it was 86% between the results of isolation in BGM cell culture and Vero cell culture and only 65.5% between the results of isolation in BGM cell culture and McCoy cell culture. For dead bird species, chlamydiosis could be diagnosed more often using DIF on smears than with isolation. The concordance between the results of the DIF on smears and isolation followed by DIF was 91%.     

防治奶牛乳房炎中药组方的筛选及其抑菌杀菌作用研究

试验根据中兽医治疗奶牛乳房炎的辨证施治原则,筛选出抑制引起奶牛乳房炎主要病原菌（金黄色葡萄球菌和大肠杆菌）的最佳中药组方,为其临床防治提供新的途径与方法。选用蒲公英、马齿苋、金银花、紫花地丁、赤芍、白芍、菊花、黄芩、漏芦、红花10味中药,通过L₁₂（2¹¹）正交试验初步筛选抗菌中药组方中的最佳优选因素,再通过L₁₆（4⁵）正交试验进一步筛选药物组方配比;采用牛津杯法测定中药组方对奶牛乳房炎主要致病菌的体外抑菌效果;采用二倍稀释法和平板计数法分别测定中药组方对奶牛乳房炎主要致病菌最小抑菌浓度（MIC）和最小杀菌浓度（MBC）。结果显示,通过L₁₂（2¹¹）正交试验初步筛选出了抗菌中药组方最佳优选因素：赤芍、黄芩、红花、菊花和白芍;L₁₆（4⁵）正交试验进一步筛选药物组方最佳配比为黄芩：赤芍：菊花：白芍：红花=3：3：3：1：2;新药组方对大肠杆菌的抑菌直径为17.39 mm,MIC和MBC分别为62.50、125.00 mg/mL,对金黄色葡萄球菌的抑菌直径为25.44 mm,MIC和MBC均为31.25 mg/mL。本研究成功筛选出了一种对奶牛乳房炎主要致病菌具有良好抑菌、杀菌作用的中药新配比组方。     

The detection of bovine lactoferrin binding protein on Toxoplasma gondii

Lactoferrin (LF), a member of the transferrin (TF) protein family, is an iron-binding protein that is known to interact with bacteria through a specific receptor. We examined the binding of bovine LF (bLF), bovine TF (bTF), and ovotransferrin (OTF) by Toxoplasma gondii using a fluorescence test and the streptavidin-biotin (SAB) method using biotin-streptavidin, and found that bLF, bTF, and OTF bound to the protein components of T. gondii. Furthermore, we confirmed that bLF, bTF, and OTF bound a 42 kDa soluble protein of T. gondii by far Western blot method. These results demonstrated that bLF binding proteins are present on T. gondii.     

Impairment of innate immune responses of airway epithelium by infection with bovine viral diarrhea virus

Bovine viral diarrhea virus (BVDV) infection is an important risk factor for development of shipping fever pneumonia in feedlot cattle, and infects but does not cause morphologic evidence of damage to airway epithelial cells. We hypothesized that BVDV predisposes to bacterial pneumonia by impairing innate immune responses in airway epithelial cells. Primary cultures of bovine tracheal epithelial cells were infected with BVDV for 48 h, then stimulated with LPS for 16 h. Expression of tracheal antimicrobial peptide (TAP) and lingual antimicrobial peptide (LAP) mRNA was measured by quantitative RT-PCR, and lactoferrin concentrations were measured in culture supernatant by ELISA. BVDV infection had no detectable effect on the constitutive expression of TAP and LAP mRNA or lactoferrin concentration in culture supernatant. LPS treatment provoked a significant increase in TAP mRNA expression and lactoferrin concentration in the culture supernatant (p<0.01), and these effects were significantly (p<0.02, p<0.01) abrogated by prior infection of the tracheal epithelial cells with the type 2 ncp-BVDV isolate. In contrast, infection with the type 1 ncp-BVDV isolate had no effect on TAP mRNA expression or lactoferrin secretion. LPS treatment induced a significant (p<0.001) upregulation of LAP mRNA expression, which was not significantly affected by prior infection with BVDV. These data indicate that infection with a type 2 BVDV isolate inhibits the LPS-induced upregulation of TAP mRNA expression and lactoferrin secretion by tracheal epithelial cells, suggesting a novel mechanism by which this virus abrogates respiratory innate immune responses and predisposes to bacterial pneumonia in cattle.     

Effects of bovine lactoferrin on in vitro replication of feline herpesvirus

Objective To investigate the effects of bovine lactoferrin on in vitro replication of feline herpes virus (FHV‐1) and to determine at what points during viral replication these effects occur. Sample population Cultured Crandell‐Reese feline kidney (CRFK) cells and FHV‐1 strain 727. Procedure Five concentrations of bovine lactoferrin (0.5, 1, 2, 5, and 10 mg/mL) were added at one or more of three time points during conventional plaque reduction assays: (a) uninfected CRFK cells were incubated in lactoferrin‐containing medium for 30 min prior to viral adsorption; (b) virus was suspended in lactoferrin‐containing medium prior to and during adsorption, or (c) CRFK cells were incubated with lactoferrin‐containing medium for 48 h following viral adsorption. Plaques were counted and antiviral effect expressed as percent inhibition relative to control medium that contained no lactoferrin. Results Exposure of CRFK cells to lactoferrin prior to or during viral adsorption inhibited FHV‐1 replication by 87–96% (mean: 91%). Application of lactoferrin following viral adsorption had no appreciable effect on FHV‐1 replication. No additive or synergistic effects were noted when lactoferrin was added at multiple steps. These effects were similar at all concentrations of lactoferrin tested. Cytotoxic effects of lactoferrin on CRFK cells were not observed at any concentration tested. Conclusions and clinical relevance Bovine lactoferrin has a notable inhibitory effect on the in vitro replication of FHV‐1 prior to and during, but not following viral adsorption. These findings strongly suggest that lactoferrin inhibits FHV‐1 adsorption to the cell surface and/or penetration of the virus into the cell. Clinical effects of topical lactoferrin in acute or recrudescent herpetic episodes in cats warrant investigation.     

Entry and survival of Salmonella enterica serotype Enteritidis PT4 in chicken macrophage and lymphocyte cell lines

Various leukocytes are involved in the reaction to counter Salmonella infection in chicken. The various leukocyte types react differently after an infection, since some clear the infection while others may cause dissemination of Salmonella throughout the chicken. Therefore, we investigated in vitro the entry and survival of Salmonella enterica serotype Enteritidis in chicken cell lines of various cell types, including two macrophage cell lines, HD11 and MQ-NCSU (NCSU), two B-cell lines LSCC-1104-X5 (1104) and LSCC-RP9 (RP9), and a T-cell line MDCC-MSB-1 (MSB-1). The macrophages were able to internalize high numbers of S. Enteritidis. In contrast and as expected, cells of the T-cell line MSB-1 and the B-cell line RP9 internalized bacteria at a much lower level. After S. Enteritidis entered the macrophages, the number of intracellular S. Enteritidis decreased over time, so that after 48h no more than 20% of the bacteria, which had entered, survived intracellularly. In contrast to macrophages, the number of S. Enteritidis in cells of the T-cell line MSB-1 and the B-cell line RP9 increased rapidly within 12h post-inoculation. Thereafter the number of intracellular S. Enteritidis decreased only slowly. In conclusion, all three different cell types were able to control and to start clearing S. Enteritidis, although macrophages were far more effective compared to T- and B-cells. However, none of the cell lines were able to clear S. Enteritidis fully within 48h. These results suggest that the three cell types play an important but different role in the dissemination and elimination of S. Enteritidis throughout the animal.     

Effect of bovine lactoferrin on functions of activated feline peripheral blood mononuclear cells during chronic feline immunodeficiency virus infection

Feline immunodeficiency virus (FIV) infection is characterized by chronic overactivation of immune and inflammatory system, resulting in anergic state and dysfunction of immune cells. Lactoferrin (LF), a glycoprotein present in exocrine secretions and neutrophils, plays an important role in host defense system. Our previous study showed that oral administration of bovine LF (bLF) suppressed oral inflammation, improved the clinical symptoms and decreased serum gamma-globulin as a marker of inflammation in FIV-infected cats with intractable stomatitis. The anti-inflammatory effect was partly involved in regulation of neutrophil function by bLF. In this study, to clarify the relationship between anti-inflammatory effects of bLF and peripheral blood mononuclear cells (PBMC), we examined the effect of bLF on proliferation, cell cycle progression and cytokine expression in mitogen-activated PBMC. MTT [3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyl tetrazolium bromide] assay showed that bLF inhibited the concanavalin A (ConA)-induced cell proliferation in FIV-infected cats with the asymptomatic carrier and AIDS-related complex (ARC) phase. Bovine LF restored ConA-induced cell cycle progression and resulted in suppression of the induced apoptosis in feline PBMC. Real-time RT-PCR showed that bLF suppressed ConA-induced expression of interferon-gamma and interleukin-2 in cells of the ARC group regardless of the time of its addition to the medium. These results suggest the hypothesis that therapy with bLF may have the potential to improve and protect functions of overactivated lymphocytes by modulating the cell proliferation, cell cycle and cytokines expression in cats in terminal stage of FIV infection.     

The detection of bovine lactoferrin binding protein on Trypanosoma brucei

Trypanosoma brucei, the causative agent of sleeping sickness in humans, requires transferrin (TF) for growth. Therefore, T. brucei has a TF receptor that allows it to obtain iron from TF. Lactoferrin (LF), a member of the TF family protein, is an iron-binding protein that is found in most biological fluids of mammals. LF has been shown to interact with some bacteria species by specific receptor-ligand binding. We examined the ability of T. brucei to bind bovine LF (bLF) by using a fluorescence test, streptavidin-biotin (SAB) microplate analysis, and far Western blotting using a biotin-streptavidin system. We found that bLF bound to components of T. brucei, and that bLF hydrolysate disrupted the sites responsible for binding to parasite proteins. Furthermore, bLF, human LF, bovine TF, and ovotransferrin bound same proteins of T. brucei, which exhibited molecular masses of 40 and 43 kDa. The N-terminal amino acid sequence of the 40 kDa bLF binding protein was identified as glyceraldehyde-3-phosphate dehydrogenase (GAPDH).     

Lactoferrin against Staphylococcus aureus Mastitis. Lactoferrin alone or in combination with penicillin G on bovine polymorphonuclear function and mammary epithelial cells colonisation by Staphylococcus aureus

Antibiotics should combine good antibacterial activity and the capacity to work in association with the host defence system. In this study, we have investigated the effects of bovine lactoferrin alone or in combination with penicillin G on the phagocytic activity of bovine polymorphonuclear leukocytes against Staphylococcus aureus. We have shown that susceptibility of S. aureus to phagocytosis was decreased in the presence of penicillin in the medium. In a kinetic study, lactoferrin alone did not affect phagocytosis but, when used with penicillin, it reversed the negative effect of this antibiotic on phagocytosis. In addition, in an epithelial invasion assay, lactoferrin alone or in combination with penicillin reduced the invasion of mammary epithelial cells in culture by S. aureus. Lactating female CD-1 mice were infected by intra-mammary delivery of a virulent penicillin-susceptible S. aureus strain and were then randomly assigned to treatments according to a 2 x 2 factorial design. In this mouse mastitis model, 2 days of systemic treatments with lactoferrin and/or penicillin did not lead to a total clearance of infection by S. aureus, but bacterial number was significantly reduced by treatments with lactoferrin or penicillin. These data suggest that bovine lactoferrin, alone or in combination with penicillin G, enhances S. aureus susceptibility to immuno-defense mechanisms, which can be beneficial in the treatment of S. aureus infections.     

Multifactorial relationships between intramammary invasion by Staphylococcus aureus and bovine leukocyte markers

We explored the hypothesis that the outcome of bacterial invasion (infection or no infection) may depend on immunologic factors when bacterial and environmental factors are kept constant. Leukocyte surface molecules (CD3, CD2, CD4, CD8, CD11b, and CD45r) were assessed before and 3 times after intramammary infusion of Staphylococcus aureus in 5 dairy cows. The somatic cell count (SCC/mL), bacterial count (colony-forming units [CFUs]/mL), ratio of milk phagocytes (mononuclear [Mphi] plus polymorphonuclear [PMN] cells) to lymphocytes (P/L index), and ratio of PMN to Mphi cells (PMN/Mphi index) were determined. Although all cows showed evidence of inflammation resulting from the infusion (the median P/L ratio was 11 times greater 1 d after infusion than before infusion), bacteria were not obtained from the milk of 2 cows. Threshold-like responses, resulting in bacterial counts that approached zero (indicating no infection) and SCCs of less than 500000/mL, were observed when the milk CD2+ lymphocyte proportion exceeded 73% (P < or = 0.007). At 1 d after infusion, 7 immune factors distinguished infected cows from those without infection with more than 95% confidence: compared with infected cows, uninfected cows had higher proportions of CD3+, CD2+, CD4+, and CD8+ T cells, higher densities of CD3 and CD2 molecules per cell, and a higher density of CD11b molecules on milk Mphi cells. At 7 d after infusion, the PMN/Mphi index was lower (94% confidence) in uninfected than in infected cows. At 14 d, the CD2, CD8, and CD45r marker densities were lower than those at 1 d (P < 0.02), findings compatible with memory function. Synergism was suggested by the combined effects of the proportions of CD3+, CD2+, and CD11b+ cells, which explained 75.5% of the bacterial-count variability (P < 0.001); alone, none of these markers predicted CFU variability. These results support further studies aimed at identifying cows capable (or incapable) of early bacterial clearance.