Study of the immunological profile towards Mycobacterium bovis antigens in naturally infected cattle

A number of studies have determined the contribution of Th1 and Th2 responses to the protective immunity and pathology of Mycobacterium bovis infection. However, much of that information is derived from experimentally infecting cattle with M. bovis and few data from naturally infected animals are available. The aim of this study was to characterize the immunological profile towards M. bovis antigens of naturally infected cattle by measurement of cytokine mRNA expression in PBMC, and to determine which lymphocyte subsets are involved in recall responses of PBMC from M. bovis infected cattle to M. bovis antigens. Consistent with data from cattle experimentally infected with M. bovis , naturally infected animals were found to display a Th1 cytokine profile in response to M. bovis PPDB stimulation. Production of IFN-γ mRNA by PBMC after PPDB stimulation statistically distinguishes between infected and healthy herds, suggesting that this molecule is usable as an M. bovis -infection marker. As happens in experimentally infected cows, CD4, CD8 and γδTCR cells from a herd naturally infected with M . bovis are the predominant T cell subsets expanded in response to PPDB.     

Étude Histopathologique Des Lésions Induites Chez La Souris Par Des Injections Intrapéritoneales Et Intracérébrales De Patuline

The injection to mice, by intraperitoneal way, of purified patulin, extracted from culture filtrates of a toxigenic strain ofAspergillus clavatus, induces symptoms similar to those observed in naturally intoxicated cattle by ingestion of forage plantlets naturally infected by the fungus. The histopathological observation shows congestives lesions in miscellaneous organs, neuronal degeneracy at the level of cerebral cortex. A direct action of patulin on the nervous centers seems not to be involved: the intracerebral injection of 0.2 mg of toxin does not produce abnormal symptoms in treated animals.     

Morphological changes of Babesia gibsoni grown in canine red blood cell-substituted severe combined immune deficiency mice

Canine red blood cell-substituted severe combined immune deficiency (Ca-RBC-SCID) mice were prepared for canine Babesia gibsoni infection. The Ca-RBC-SCID mice infected with B. gibsoni developed a high level of parasitemia, and showed clinical symptoms such as anemia and hemoglobinuria, which are similar to those observed in dogs infected with B. gibsoni. The B. gibsoni parasites grown in Ca-RBC-SCID mice showed marked morphological changes, including a significantly larger size of parasites than those in dogs and abundant RBCs containing 4, 8, 16, and 32 parasites. The multiple infection may have resulted from 1 parasite because the posterior end of each parasite in a multiply infected cell was connected. The parasites grown in SCID mice retained their infectivity and virulence to dogs and their morphology was dramatically restored to the original state when they were returned to dogs.     

Immune responses to the enduring hypoxic response antigen Rv0188 are preferentially detected in Mycobacterium bovis infected cattle with low pathology

The DosR regulon and the Enduring Hypoxic Response (EHR) define a group of M. tuberculosis genes that are specifically induced in bacilli exposed in vitro to conditions thought to mimic the environment encountered by Mycobacteria during latent infection. Although well described in humans, latent mycobacterial infection in cattle remains poorly understood. Thus, the aim of this study was to identify antigens that may potentially disclose cattle with latent M. bovis infection. To this end, we initially screened 57 pools of overlapping peptides representing 4 DosR regulon and 29 EHR antigens for their ability to stimulate an immune response in whole blood from TB-reactor cattle using IFN-γ and IL-2 as readouts. All 4 DosR regulon proteins were poorly recognized (maximum responder frequency of 10%). For the EHR antigens, both IFN-γ and IL-2 revealed similar response hierarchies, with responder frequencies ranging from 54% down to 3% depending on the given EHR antigen. Furthermore, these results demonstrated that responses in the infected cattle were largely IFN-γ biased. To support the concept for their role in latency, we evaluated if EHR antigen responses were associated with lower pathology. The EHR antigen Rv0188 was recognised predominantly in animals presenting with low pathology scores, whereas responses to ESAT-6/CFP-10 or the other EHR antigens tested were prevalent across the pathology spectrum. However, when we determined the production of additional cytokines induced by the M. bovis antigens PPD-B or ESAT-6/CFP-10, we detected significantly greater PPD-B-induced production of the pro-inflammatory cytokine IL-1β in animals recognizing Rv0188 (i.e. those with limited or no pathology). Thus, these results are consistent with the idea that responses to Rv0188 may identify a subset of animals at early stages of infection or in which disease progression may be limited.     

New insights into the altered adhesive and mechanical properties of red blood cells parasitized by Babesia bovis

Sequestration of parasite-infected red blood cells (RBCs) in the microvasculature is an important pathological feature of both bovine babesiosis caused by Babesia bovis and human malaria caused by Plasmodium falciparum. Surprisingly, when compared with malaria, the cellular and molecular mechanisms that underlie this abnormal circulatory behaviour for RBCs infected with B. bovis have been relatively ignored. Here, we present some novel insights into the adhesive and mechanical changes that occur in B. bovis-infected bovine RBCs and compare them with the alterations that occur in human RBCs infected with P. falciparum. After infection with B. bovis, bovine RBCs become rigid and adhere to vascular endothelial cells under conditions of physiologically relevant flow. These alterations are accompanied by the appearance of ridge-like structures on the RBC surface that are analogous, but morphologically and biochemically different, to the knob-like structures on the surface of human RBCs infected with P. falciparum. Importantly, albeit for a limited number of parasite lines examined here, the extent of these cellular and rheological changes appear to be related to parasite virulence. Future investigations to identify the precise molecular composition of ridges and the proteins that mediate adhesion will provide important insight into the pathogenesis of both babesiosis and malaria.     

Can Babesia infections be used as a model for cerebral malaria?

Infections with certain species of Plasmodium and Babesia induce, among other symptoms, cerebral pathology. The finding of heavily parasitized cerebral capillaries upon postmortem examination has led to the assumption that blockage of capillaries with infected red blood cells caused the cerebral symptoms and subsequent death. As this type of cerebrovascular pathology is found both in humans dying from malaria and in cattle dying from babesiosis, the latter could possibly be used as an animal model for the study of human cerebral malaria. However, before such a model system is adopted, the experimental data concerning cerebral pathology of babesiosis needs critical evaluation. Here, Theo Schetters and Wijnand Eling review the pathological mechanisms in cerebral babesiosis and relate these to cerebral malaria. Finally, they discuss the use of animal model systems for specific aspects of the pathological picture.     

PCR-based detection of Babesia bovis and Babesia bigemina in their natural host Boophilus microplus and cattle

PCR and nested-PCR methods were used to assess the frequency of Babesia bovis and Babesia bigemina infection in Boophilus microplus engorged females and eggs and in cattle reared in an area with endemic babesiosis. Blood and the engorged female ticks were from 27 naturally infested calves and 25 crossbred cows. The frequency of both Babesia species was similar in calves and cows (P>0.05). Babesia bovis was detected in 23 (85.2%) calves and in 25 (100%) cows and B. bigemina was detected in 25 (92.6%) calves and in 21 (84%) cows. Mixed infections with the both Babesia species were identified in 42 animals, 21 in each age category. Of female ticks engorged on calves, 34.9% were negative and single species infection with B. bigemina (56.2%) was significantly more frequent (P<0.01) than with B. bovis (4.7%). Most of the females (60.8%) engorged on cows did not show Babesia spp. infection and the frequency of single B. bovis infection (17.6%) was similar (P>0.05) to the frequency of single B. bigemina infection (15.9%). Mixed Babesia infection was lower (P<0.01) than single species infection in female ticks engorged either in cows (5.7%) or in calves (4.3%). An egg sample from each female was analysed for the presence of Babesia species. Of the egg samples from female ticks infected with B. bovis, 26 (47.3%) were infected while from those from female ticks infected with B. bigemina 141 (76.6%) were infected (P<0.01). The results showed that although the frequency of both species of Babesia was similar in calves and cows, the infectivity of B. bigemina was higher to ticks fed on calves while to those ticks fed on cows the infectivity of both Babesia species was similar.     

Babesia bovis: subcellular localization of host erythrocyte membrane components during their asexual growth

In the present study, the subcellular localization of the host red blood cell (RBC) membrane components, the alpha2-3-linked sialic acid (SA) residues and the lipid bilayer, was observed during the asexual growth of Babesia bovis using two erythrocyte probes, the SA-specific lectin (MALII) and the lipophilic fluorescent (PKH2) probes, respectively. In confocal laser scanning microscopy with MALII, the SA residues on the surface of parasitized RBCs appeared to accumulate into the intracellular parasites as the parasites matured as well as to remain on the surface of extracellular parasites. Furthermore, when PKH2-labeled RBCs were infected with B. bovis, PKH2 signals were also observed around both the intracellular and the extracellular parasites, similarly to the results of MALII. These results indicated that the components derived from the host erythrocyte membrane are incorporated into the intracellular parasites during their asexual growth within the parasitized RBC, suggesting the possible formation of a parasitophorous vacuole-based network or a parasite surface coat.     

Heparin binding haemagglutin as potential diagnostic marker of Mycobacterium bovis

In this study, we characterized the humoral responses in cattle of Sardinia. The animals were divided into three groups: 1) 28 cattle infected with Mycobacterium bovis; 2) 48 cattle from herds in which foci of infection was notified; 3) 50 cattle from herds that were TB-free. Levels of IgG antibody were measured against the following antigens of M. tuberculosis: Heparin-Binding-Haemagglutin (HBHA), Ag85B, PPE44, and PE_PGRS33 to investigate their potential to diagnose TB in animals. Our results indicated that HBHA is a potential candidate for the development of a serological assay for rapid diagnosis of cattle infected with M. bovis.     

小鼠感染合胞病毒与鼠流感病毒后肺脏病理学比较研究

目的与方法用合胞病毒(RSV)和鼠肺流感病毒(IVP)感染SPF级BALB/c小鼠,复制两种不同的小鼠病毒性肺炎动物模型,观察其临床症状,并对两种模型各自的肺部组织病理学特点进行研究。结果与结论IVP模型组与RSV模型组小鼠感染病毒后,分别在试验的2天和3天发病,临床症状均表现为精神沉郁、耸毛、卷缩、毛无光泽、活动减少、呼吸急促、咳嗽。但IVP感染模型组小鼠在发病后出现死亡,第7天其死亡率达到40%,而RSV感染模型组小鼠7日试验内无死亡病例发生。病理组织学诊断,RSV模型组小鼠为急性渗出性间质性肺炎,IVP模型组小鼠为出血性间质性肺炎。     

Cloning, sequencing, and expression of interferon-gamma from elk in North America

Eradication of Mycobacterium bovis relies on accurate detection of infected animals, including potential domestic and wildlife reservoirs. Available diagnostic tests lack the sensitivity and specificity necessary for accurate detection, particularly in infected wildlife populations. Recently, an in vitro diagnostic test for cattle which measures plasma interferon-gamma (IFN-gamma) levels in blood following in vitro incubation with M. bovis purified protein derivative has been enveloped. This test appears to have increased sensitivity over traditional testing. Unfortunately, it does not detect IFN-gamma from Cervidae. To begin to address this problem, the IFN-gamma gene from elk (Cervus elaphus) was cloned, sequenced, expressed, and characterized. cDNA was cloned from mitogen stimulated peripheral blood mononuclear cells. The predicted amino acid (aa) sequence was compared to known sequences from cattle, sheep, goats, red deer (Cervus elaphus), humans, and mice. Biological activity of the recombinant elk IFN-gamma (rElkIFN-gamma) was confirmed in a vesicular stomatitis virus cytopathic effect reduction assay. Production of monoclonal antibodies to IFN-gamma epitopes conserved between ruminant species could provide an important tool for the development of reliable, practical diagnostic assays for detection of a delayed type hypersensitivity response to a variety of persistent infectious agents in ruminants, including M. bovis and Brucella abortus. Moreover, development of these reagents will aid investigators in studies to explore immunological responses of elk that are associated with resistance to infectious diseases.     

Multimodal analysis of Plasmodium knowlesi‐infected erythrocytes reveals large invaginations,swelling of the host cell,and rheological defects

The simian parasite Plasmodium knowlesi causes severe and fatal malaria infections in humans, but the process of host cell remodelling that underpins the pathology of this zoonotic parasite is only poorly understood. We have used serial block‐face scanning electron microscopy to explore the topography of P. knowlesi‐infected red blood cells (RBCs) at different stages of asexual development. The parasite elaborates large flattened cisternae (Sinton Mulligan's clefts) and tubular vesicles in the host cell cytoplasm, as well as parasitophorous vacuole membrane bulges and blebs, and caveolar structures at the RBC membrane. Large invaginations of host RBC cytoplasm are formed early in development, both from classical cytostomal structures and from larger stabilised pores. Although degradation of haemoglobin is observed in multiple disconnected digestive vacuoles, the persistence of large invaginations during development suggests inefficient consumption of the host cell cytoplasm. The parasite eventually occupies ~40% of the host RBC volume, inducing a 20% increase in volume of the host RBC and an 11% decrease in the surface area to volume ratio, which collectively decreases the ability of the P. knowlesi‐infected RBCs to enter small capillaries of a human erythrocyte microchannel analyser. Ektacytometry reveals a markedly decreased deformability, whereas correlative light microscopy/scanning electron microscopy and python‐based skeleton analysis (Skan) reveal modifications to the surface of infected RBCs that underpin these physical changes. We show that P. knowlesi‐infected RBCs are refractory to treatment with sorbitol lysis but are hypersensitive to hypotonic lysis. The observed physical changes in the host RBCs may underpin the pathology observed in patients infected with P. knowlesi.     

Bovine babesiosis: failure to induce interferon gamma production in response to Babesia bovis antigens in cattle.

The immune response to Babesia bovis infection or vaccination was evaluated by measuring antibody and interferon gamma (IFN-gamma) production to protective recombinant and crude native B. bovis antigens. Cells from vaccinated or infected cattle failed to produce detectable IFN-gamma when stimulated with B. bovis antigens in vitro. In contrast, antibody was induced by protective recombinant B. bovis antigens. These findings are consistent with the argument that immunity to B. bovis infection is correlated most strongly with humoral rather than cell-mediated immune responses.     

Experimental inoculation of meadow voles (Microtus pennsylvanicus), house mice (Mus musculus), and Norway rats (Rattus norvegicus) with Mycobacterium bovis

Mycobacterium bovis has a wide host range that includes several wildlife species, and this can hamper attempts to eradicate bovine tuberculosis from livestock. The purpose of this study was to determine if common rodent species, namely meadow voles (Microtus pennsylvanicus), house mice (Mus musculus), and Norway rats (Rattus norvegicus), that inhabit the bovine tuberculosis endemic area of Michigan, can be experimentally infected with M. bovis. The objectives of the study were: 1) to determine if these rodent species can be infected, and if so, to document attendant pathologic processes/pathogenesis; 2) to detect any fecal shedding of M. bovis; and 3) to evaluate the relative susceptibility of the three species to M. bovis infection. For each species (n=36) there were two treatment (n=12/group) and one or two control groups depending on species (n=6-12/group); the maximum study duration was 60 days. The meadow vole treatments consisted of high dose inocula that were given by oral or intranasal routes, whereas the house mice and Norway rats were given only oral inocula at either a high or low dose. Of the three species, meadow voles were most susceptible to M. bovis infection. Upon intranasal inoculation, all 12 voles were infected as determined by gross and microscopic lesions and culture of M. bovis from tissue and feces. Seven of the 12 meadow voles inoculated orally were infected. House mice also were susceptible; M. bovis was isolated from 14 of 24 animals. Only one Norway rat in the high dose treatment group was positive by culture and this was the only animal from which minimal attendant lesions were observed. Results of this study indicate that meadow voles and house mice can be infected with M. bovis and might serve as spillover hosts. Concerted efforts should, therefore, be made to reduce or eliminate these rodents on premises where M. bovis-infected livestock are present.     

Helminth parasites of intermingling axis deer, wild swine and domestic cattle from the island of Molokai, Hawaii

Helminth infections of axis deer (Cervus axis), wild swine (Sus scrofa) and domestic cattle (Bos taurus) were studied among intermingling herds on the Puu-O-Hoku Ranch, Molokai, Hawaii. Twenty-four species of helminths were collected from the 10 deer, 10 swine and 10 cattle. Capillaria bovis, Cooperia punctata, Haemonchus contortus and Trichostrongylus axei infected both axis deer and cattle, whereas Gongylonema pulchrum infected both axis deer and wild swine. None of the species of helminths occurred in both wild swine and cattle nor was any species found in all three hosts. Wild swine and domestic cattle supported separate and distinct helminth communities. In contrast, the helminth community of axis deer appeared to be derived from the helminth communities of cattle and wild swine and consisted only of those species capable of parasitizing either a broad range of ruminants or many mammalian taxa.     

Antigen recognition and immunomodulation by gamma delta T cells in bovine tuberculosis

This report describes the in vitro proliferative responses of peripheral blood gammadelta T cells to defined mycobacterial protein Ags and the immunomodulatory effect of gammadelta T cells in cattle infected with Mycobacterium bovis. gammadelta T cell responses were specific to M. bovis infection because they were detected in cattle either experimentally or naturally infected with M. bovis, but were not present in uninfected controls. Proliferating gammadelta T cell cultures produced enhanced levels of IFN-gamma and TGF-beta, but not IL-2 in response to the more immunodominant mycobacterial AGS: Depletion of gammadelta T cells from PBMC resulted in an increased Ag-specific proliferation in half the animals tested, indicating a suppressive effect of gammadelta T cells upon other (alphabeta) T cell responses. Because gammadelta T cells constitute a major T cell population in the peripheral blood of cattle, the activities of gammadelta T cells described in this report could make a significant contribution to the immune response in bovine tuberculosis.     

Tick-borne diseases in ruminants of Central and Southern Italy: epidemiology and case reports

Sera and blood from cattle and sheep were examined for the presence of Babesia and Theileria spp by microscopy and serology at the Parasitology Department of the Istituto Zooprofilattico Sperimentale of Abruzzo and Molise (IZSAM). Of the 47 bovine herds (323 animals) tested, 15 were found positive for Babesia bigemina and 1 for Babesia bovis. Two outbreaks occurred, one caused by B. bigemina and one by B. bovis. The B. bigemina outbreak occurred in Abruzzo and has been followed for two years. The isolate of B. bigemina was very pathogenic leading to the death of two cows out of 57. The vector responsible of the transmission appeared to be Rhipicephalus bursa. Parasites were observed in the erythrocytes for 30 days whereas sera were positive to indirect immunofluorescence (IFA) for at least one year. The B. bovis outbreak occurred in the province of Mantova (Northern Italy) in a group of 70 beef cattle imported from France. The infection resulted in the death of 5 animals and severe illness in another 6. In contrast with what occurred for Babesia infection, no clinical cases were recorded in cattle when species of Theileria were detected by microscopy. Of the 24 bovine herds (252 animals) tested for Theileria, 21 were found positive for the T. "sergenti"/buffeli/orientalis group. Single and mixed infection of T. "sergenti" and T. buffeli/orientalis were detected in herds of cross-bred cattle from Abruzzo and Marche. The parasites were identified by using a polymerase chain reaction which amplified DNA encoding p32/34. Most of the collected ticks (90%) were adults of R. bursa whereas the others were adults of Hyalomma detritum. During the period the animals have been observed (18 months), no clinical cases have been recorded and no associations have been found between blood abnormalities and animals found infected with Theileria. Prevalences of subclinically infected carriers increased from February till December (95.4%) even if the animals were indoors and no ticks were present. The prevalence then dropped dramatically six months later (76.7%). In calves less than 1 year old, the prevalence of infection significantly (p<0.05) increased with age, however intraerythrocytic stages of Theileria were found in the blood of three newborn calves (<7 days of age). Of the 18 ovine flocks tested for Babesia spp. (150 animals examined), 1 was positive for B. ovis and 2 for B. motasi. B. motasi infection was not associated with symptoms, while an outbreak of babesiosis caused by B. ovis occurred in Abruzzo. The infection resulted in the death of 3 animals (0.75% of the flock), two rams (20% of the total number) and a ewe, and severe illness in another 5 ewes (2% of the flock). Specimens of R. bursa and R. turanicus were collected from the infected animals. Of the 18 flocks (150 animals) examined, 12 were microscopically positive for Theileria spp. No clinical cases were recorded and identification at species level was not possible on the basis of morphological criteria. The prevalence distribution of infected herds and infected animals within herds and flocks have been calculated by a Monte Carlo simulation model, running 10,000 iterations. The most likely levels of prevalence of infected herds and infected animals within herds found for the species observed were as follows: 20% for B. bigemina with a prevalence within herd of 27%, 11% for B. bovis (18% within herd), 10% for Babesia ovis (19% within herd), 10% for B. motasi (17.5% within herd), 63% for Theileria in cattle (66% within herd) and 51% for Theileria in sheep (55% within herd).     

Is Mycobacterium bovis in the environment important for the persistence of bovine tuberculosis?

Mycobacterium bovis is the causative agent of bovine tuberculosis (bTB) in cattle and wildlife. Direct aerosol contact is thought to be the primary route of infection between conspecifics, whereas indirect transmission via an environmental reservoir of M. bovis is generally perceived not to be a significant source for infection. Here, we report on the application of molecular technology (PCR) to quantify the prevalence of M. bovis in the environment and to explore its epidemiological significance. We show that the detectability of viable M. bovis at badger setts and latrines is strongly linked to the frequency of M. bovis excretion by infected badgers, and that putative M. bovis in the environment is prevalent on a large proportion of endemic cattle farms in Britain. These results raise important questions about the role of an environmental reservoir in bTB persistence.     

Virulent and Avirulent Strains of Babesia bovis: The Relationship Between Parasite Protease Content and Pathophysiological Effect of the Strain

A virulent strain of Babesia bovis (“L” strain) was rendered avirulent by irradiation with 35 krads with a γ source. Another virulent strain of B. bovis (“C” strain) was made avirulent by rapid blood passage through 12 splenectomised calves. Both the parent virulent and their respective avirulent strains were injected into susceptible cattle. A nonfatal disease was observed in those intact cattle that had avirulent parasites; however, a fatal disease was produced in those animals that had received virulent parasites and in splenectomised calves that had received avirulent parasites. Blood kinin levels rose and plasma kininogen levels fell significantly in those animals infected with both virulent strains. Nonsignificant changes occurred with these parameters in animals infected with avirulent parasites. Preparations of disrupted parasites were obtained from the four parasite populations. Both virulent strains contained high levels of protease. The avirulent forms contained insignificant amounts. As parasite doubling times and maximum parasitaemias were the same for all four parasite populations, we conclude that these enzymes are not obligatory for parasite multiplication in the vertebrate host. Their role in producing pathological changes in the host is discussed.     

Development of vaccines to control bovine tuberculosis in cattle and relationship to vaccine development for other intracellular pathogens

Vaccination of cattle against bovine tuberculosis could be an important strategy for the control of disease either where there is a wildlife reservoir of Mycobacterium bovis infection or in developing countries where it is not economically feasible to implement a 'test and slaughter' control program. Advances in the understanding of protective immune responses to M. bovis infection in cattle and the advent of new molecular biological techniques, coupled with the sequencing of the M. bovis genome have provided opportunities for the rational development of improved tuberculosis vaccines. A number of new tuberculosis vaccines including attenuated M. bovis strains, killed mycobacteria, protein and DNA vaccines are under development and many are being assessed in cattle. Recent results have revealed several promising vaccine candidates and vaccination strategies. Ways of distinguishing between vaccinated and infected cattle are becoming available and the possibility of new approaches to the eradication of tuberculosis from domestic livestock is discussed. Similarities between the mechanisms of protective immunity against M. bovis and against other intracellular parasites continue to be found and discoveries from vaccine studies on bovine tuberculosis may provide helpful insights into requirements for vaccines against other intracellular pathogens.