Epidemiology of bovine venereal campylobacteriosis: geographic distribution and recent advances in molecular diagnostic techniques.

Bovine venereal campylobacteriosis (BVC) is a major cause of economic loss to the cattle industries in different parts of the world. Camplylobacter fetus subsp. venerealis (Cfv), the main causative agent of BVC, is highly adapted to the genital tract of cattle and is transmitted by carrier bulls. However, infertility and abortions can also be caused by the intestinal pathogens C. fetus subsp. fetus (Cff), and C. jenuni, which are not venereally transmitted. Bovine venereal campylobacteriosis, caused by Cfv associated with lowered fertility, embryo mortality and abortion, repeated returns to service, reduced pregnancy rates and extended calving intervals, has the highest prevalence in developing countries where natural breeding in cattle is widely practised. The epidemiology, pathogenesis and diagnosis of the disease have been the subject of previous reviews. The main focus of this review is to highlight the epidemiology of this disease with particular reference to geographical distribution and recent advances in molecular diagnostic techniques. It is hoped that further research interest of scientists will be stimulated with a view to finding lasting solutions to the reproductive problems associated with the disease for better livestock productivity, particularly in developing endemic countries.

Relative importance of Ixodes ricinus and Ixodes trianguliceps as vectors for Anaplasma phagocytophilum and Babesia microti in field vole (Microtus agrestis) populations.

The importance of Ixodes ricinus in the transmission of tick-borne pathogens is well recognized in the United Kingdom and across Europe. However, the role of coexisting Ixodes species, such as the widely distributed species Ixodes trianguliceps, as alternative vectors for these pathogens has received little attention. This study aimed to assess the relative importance of I. ricinus and I. trianguliceps in the transmission of Anaplasma phagocytophilum and Babesia microti among United Kingdom field voles (Microtus agrestis), which serve as reservoir hosts for both pathogens. While all instars of I. trianguliceps feed exclusively on small mammals, I. ricinus adults feed primarily on larger hosts such as deer. The abundance of both tick species and pathogen infection prevalence in field voles were monitored at sites surrounded with fencing that excluded deer and at sites where deer were free to roam. As expected, fencing significantly reduced the larval burden of I. ricinus on field voles and the abundance of questing nymphs, but the larval burden of I. trianguliceps was not significantly affected. The prevalence of A. phagocytophilum and B. microti infections was not significantly affected by the presence of fencing, suggesting that I. trianguliceps is their principal vector. The prevalence of nymphal and adult ticks on field voles was also unaffected, indicating that relatively few non-larval I. ricinus ticks feed upon field voles. This study provides compelling evidence for the importance of I. trianguliceps in maintaining these enzootic tick-borne infections, while highlighting the potential for such infections to escape into alternative hosts via I. ricinus.

The immune status of the bovine uterus during the peripartum period.

The post-partum period in cattle is characterised by an increased risk of infection of the uterus, as the anatomical barriers are broached during parturition and remain open for several days. Infection of the uterus is largely influenced by the balance between bacterial contamination and the local and systemic immune status during pregnancy and around parturition. Infectious diseases are more prevalent during this period, because of an impaired immune status before and immediately after parturition. Neutrophils play a primary role in the defence of the uterus against infection. Influx of neutrophils into the uterus is thought to be mediated by chemoattractants, chemokines and adhesion molecules, such as beta2-integrin (complement receptor 3) and L-selectin (CD62L). Other cellular components activated in the uterus during this period include lymphocytes, eosinophils, mast cells and macrophages. The major classes of immunoglobulins (IgM, IgA and IgG), either by passive diffusion or local production, play an important protective role in the uterus by acting as opsonins to enhance phagocytosis, stimulating the complement pathways or blocking pathogens from adhering to mucosal surfaces. Endometrial cells express toll-like receptor 4 (TLR4), which recognises lipopolysaccharides of Escherichia coli and other Gram negative bacteria, the most common causes of bovine endometritis. Activation of TLR4 triggers the production of tumour necrosis factor alpha and other pro-inflammatory cytokines. The periparturient period is also characterised by an increased secretion of prostaglandin F(2alpha), which enhances uterine immune defences.

Sympatric Ixodes trianguliceps and Ixodes ricinus ticks feeding on field voles (Microtus agrestis): potential for increased risk of Anaplasma phagocytophilum in the United Kingdom?

The importance of wild rodents as reservoirs of zoonotic tick-borne pathogens is considered low in the United Kingdom because, in studies to date, those parasitized by exophilic Ixodes ricinus ticks carry almost exclusively larvae and thus have a minor role in transmission cycles. In a cross-sectional study, 11 (6.7%) of 163 field voles (Microtus agrestis) captured at field sites in Northern England were PCR-positive for Anaplasma phagocytophilum. The voles were found to act as hosts for both larval and nymphal I. ricinus and all stages of the nidicolous tick I. trianguliceps, and eight individuals were infested with ticks of both species at the same time. Two of 158 larval and one of 13 nymphal I. ricinus, as well as one of 14 larval and one of 15 nymphal I. trianguliceps collected from the rodents were PCR-positive. These findings suggest that habitats where field voles are abundant in the United Kingdom may pose a risk of A. phagocytophilum infection because (i) field voles, the most abundant terrestrial mammal in the United Kingdom, may be a competent reservoir; (ii) the field voles are hosts for both nymphal and larval ixodid ticks so they could support endemic cycles of A. phagocytophilum; and (iii) they are hosts for nidicolous I. trianguliceps, which may alone maintain endemic cycles, and exophilic I. ricinus ticks, which could act as a bridge vector and transmit infections to humans and domesticated animals.

Detection by the polymerase chain reaction of Anaplasma phagocytophilum in tissues of persistently infected sheep.

To investigate the reservoir tissues of the tick-borne bacterium Anaplasma phagocytophilum in persistently infected sheep, six 6-month-old lambs were infected with a field isolate of the bacterium and maintained under tick-free conditions. At one and two weeks post-infection, A. phagocytophilum was detected in the peripheral blood of all lambs by examining May-Grünwald Giemsa-stained blood smears for classical intra-neutrophil inclusions, and by an A. phagocytophilum-specific nested PCR. After euthanasia at 3 months post-inoculation, peripheral blood and numerous tissue samples were collected from each lamb. DNA extracted from these samples was then subjected to PCR. All blood samples were PCR-negative but three lambs had PCR-positive tissues including intestinal wall and lymph nodes, thymus, bone marrow, kidney and bladder wall. The widespread nature of PCR-positive tissues suggested that circulatory cells may form the reservoir cells for A. phagocytophilum infection in carrier sheep, rather than lymphoid tissues as in rodents. PCR-positive tissue and blood samples were strikingly fewer in the experimentally infected sheep than reported earlier in tick-exposed carrier sheep under field conditions. It seems possible that tick infestation amplifies A. phagocytophilum infections in carrier sheep to a degree that enables tick transmission to occur.

Antigenicity of ovine strains of Anaplasma phagocytophilum grown in tick cells and ovine granulocytes.

Antigens prepared from ovine granulocytes and tick cells infected with ovine strains of Anaplasma phagocytophilum, the causative agent of tick-borne fever, were tested in respect of their suitability for the assay of antibodies in ovine sera by enzyme-linked immunosorbent assay (ELISA) and by indirect immunofluorescent antibody test (IFAT). Antigens prepared from tick cells were as sensitive and specific as those expressed in ovine granulocytes for the detection of specific antibodies by ELISA, but they failed to react in the IFAT with immune sera obtained from sheep previously infected with ovine strains of A. phagocytophilum.

Progeny of sheep persistently infected with border disease virus.

Most lambs affected with border disease die early in life but those which survive gradually loose their body tremors and their fleece abnormalities become less clear. Seven female lambs persistently infected with border disease virus were reared to maturity and bred from when they were 2 to 3 years old. Two failed to conceive but five gave birth to 6 live lambs with clinical signs of border disease characterized by hairy and pigmented fleece with or without body tremors. The epidemiological significance of persistently infected sheep is discussed.

Granulocytic ehrlichiosis: an emerging or rediscovered tick-borne disease?

The Ehrlichieae are gram-negative obligately intracellular bacterial pathogens. They can be divided into at least three genogroups on the basis of 16S rRNA gene sequences, but are also classified by target cell specificity. A group of granulocytic ehrlichiae primarily infect neutrophils and fall into genogroup II. The granulocytic ehrlichiae are subdivided by their target hosts, i.e., Ehrlichia phagocytophila in cattle and sheep, E. equi in horses, and the agents of human (HGE) and llama (LGE) granulocytic ehrlichioses. However, these subdivisions may give a false impression, as all these species are closely related both antigenically and on the basis of 16S rRNA operon sequence. In addition, cross-species transmission can occur naturally or by experimental infection. The vectors for these granulocytic ehrlichiae are hard-bodied ixodid ticks, and the reservoir hosts are probably wild rodents, deer and sheep. In each host, this illness presents as a febrile disease which can be followed by immunosuppression leading to secondary infections.

Evidence of immunosuppression by bovine respiratory syncytial virus.

Respiratory syncytial virus (RSV) is a major respiratory pathogen in human infants and calves. Calves and lambs infected with bovine RSV show mild clinical signs but they are more susceptible to secondary infection with Pasteurella haemolytica. Lambs infected with P. haemolytica 6 days after experimental infection with bovine RSV had significantly greater magnitudes of fever, higher disease and lesion scores and higher mortality rates than those infected with P. haemolytica or bovine RSV alone (P less than 0.05). Experimental infection with bovine RSV is characterized by alterations in lymphocyte subpopulations and down-regulation of some of their functions. For example, the number of T helper cells is significantly reduced during the first week of infection and peripheral blood mononuclear cells obtained from bovine RSV-infected lambs were less responsive to the mitogen phytohaemagglutinin but more susceptible to P. haemolytica cytotoxin than those obtained from control lambs. Infection with bovine RSV does not significantly affect the humoral immune responses of lambs against P. haemolytica cytotoxin. Bovine RSV does not appear to affect the capacity of alveolar macrophages to present antigens in vitro.

Lymphocyte subpopulations in peripheral blood of lambs experimentally infected with Pasteurella haemolytica.

The lymphocyte subpopulations in peripheral blood obtained from eleven lambs experimentally infected with Pasteurella haemolytica were compared with those obtained from eight control lambs by flow cytometry, using a panel of monoclonal antibodies against specific lymphocyte epitopes. Experimental infection with P. haemolytica was characterized by a transient but significant reduction in SBU-T1+ (CD5+) T cells and SBU-T4+ (CD4+ or helper) T lymphocytes (P less than 0.05) and a significant rise in lymphocytes which did not express the LCA p220 epitope and the pan T cell surface marker (CD5-LCA p220-) ("null"). The reductions in CD5+ and CD4+ lymphocytes occurred 24 h after experimental infection, returning to preinoculation levels 5 days post inoculation (DPI). Five to 9 days after experimental infection, there was a significant increase in the number of lymphocytes, which expresses the pan T cell surface marker (CD5+) but which were CD4-CD8-. Lymphocyte transformation responses to the mitogen phytohaemagglutinin (PHA) were significantly reduced 24 h after experimental infection with P. haemolytica (P less than 0.05).

Pathogenesis of bovine respiratory syncytial virus in experimentally infected lambs.

Twenty-four 6-8-week-old conventionally reared lambs were inoculated intranasally and intratracheally with bovine respiratory syncytial virus. Infected lambs showed mild clinical signs characterized by slight serous nasal discharge, coughing, lachrymation and bronchovascular sounds on the middle part of the lung 5-9 days post-inoculation (PI). Virus was isolated in nasal swabs from 9 of 24 lambs between 3 and 7 days PI. However, virus was recovered from tracheal and lung tissue of all lambs killed between 3 and 11 days PI. Virus-specific antibodies appeared as early 6 days PI but high titres were attained 14-21 days PI. Lungs of lambs killed on different days PI had multifocal areas of consolidation. There was an increase of lymphocytes with a T-suppressor cell marker and a decrease in those with a T-helper marker in lung lavages obtained 5 days PI.

Alterations in lymphocyte subpopulations in peripheral blood of sheep persistently infected with border disease virus.

Fourteen sheep persistently infected with border disease virus were investigated to examine the effects of persistent viraemia on lymphocyte subpopulations in peripheral blood and their responses to the mitogen phytohaemagglutinin (PHA) in vitro. Persistently infected sheep had significantly more CD8+ (cytotoxic-suppressor) T-lymphocytes than uninfected sheep of the same age (P less than 0.001). The total number of CD4+ (helper) T-lymphocytes were not significantly different but there were more T-lymphocytes (CD5+) which were CD4- and CD8- in normal sheep than persistently infected sheep (P less than 0.001). Peripheral lymphocytes obtained from persistently infected sheep showed significantly reduced blastogenesis induced by PHA than those obtained from normal sheep (P less than 0.001).

Lymphocyte responses to viral antigens and phytohaemagglutinin in persistently viraemic sheep and lambs experimentally infected with Border disease virus.

Lymphocytes obtained from lambs 5 to 21 days after experimental infection with Border disease virus (BDV) showed significant blastogenic responses to live or inactivated BDV. Live virus stimulated significantly higher lymphocyte transformation (LT) responses than inactivated virus. Lymphocytes obtained from uninfected control and persistently infected lambs had no significant response to live or inactivated antigen. Lymphocyte responses to phytohaemagglutinin were significantly higher in control lambs than in experimentally infected lambs in samples obtained 5 to 10 days post-inoculation.

Tick-borne fever: leucocyte migration inhibition.

When sheep were infected with Cytoecetes phagocytophila the in vitro migration of peripheral leucocytes was reduced even when antigen was not added. The inhibition of migration coincided with parasitaemia and was due to soluble factors which inhibited the migration of leucocytes from normal sheep. After the cessation of parasitaemia addition of antigen was necessary for migration inhibition to occur.

In vitro propagation of Cytoecetes phagocytophila, the causative agent of tick-borne fever.

Cytoecetes phagocytophila, a neutrophilic rickettsia which parasitizes sheep and cattle, was propagated transiently at 37 degrees C in cultures of heparinized whole blood of sheep supplemented with Medium 199 containing HEPES buffer. Significant increases in the number of infected cells and the number of rickettsias per infected cell were observed within 24 h. Back-passage into sheep produced typical tick-borne fever.

Replication of bovine respiratory syncytial virus in ovine peripheral blood lymphocytes and monocytes in vitro.

Adherent and non-adherent mononuclear cells obtained from the peripheral blood of normal lambs supported the replication of bovine respiratory syncytial virus in vitro. Sequential treatment of monocytes with phorbol ester acetate (PMA) enhanced their ability to support viral replication. After exposure in vitro for 24 h, viral antigens were present in 47 +/- 4.5% of monocytes and 32 +/- 3% of lymphocytes. Treatment of monocytes with PMA resulted in the increase of the proportion of cells expressing viral antigen and in the titre of infectious virus.

Immune responses of the camel (Camelus dromedarius) to contagious ecthyma (Orf) virus infection.

An enzyme-linked immunosorbent assay (ELISA) was developed together with a western blotting technique for the detection of total and specific IgG and IgM antibodies to the contagious ecthyma (orf) virus in camel (Camelus dromedarius) sera and for identifying the seroreactive antigens of the virus. An outbreak of generalised contagious ecthyma in camels was diagnosed for the first time in Libya; the seropositivity rate in a herd with clinically affected camels was 37.9% (and was related to clinical signs) and in apparently normal herds was 0% to 6.8%. Two viral antigenic determinants (22 and 40 kDa) were shared by the western blotting patterns of all the positive camel sera tested, another viral antigenic component of 28 kDa was shared by the positive sera with high ELISA titres. Very close similarity was seen with the western blot of orf-positive sheep sera. It is considered that the ELISA technique was valid for orf serodiagnosis in the camel and could be usefully applied to other species at risk of orf infection.

Replication of bovine respiratory syncytial virus in bovine and ovine peripheral blood lymphocytes and monocytes and monocytic cell lines.

The present study compared the replication of bovine respiratory syncytial virus (BRSV) in bovine and ovine peripheral blood mononuclear cells, ovine and bovine monocytic cell lines and ovine alveolar macrophages. Low titres of virus were detected in ovine and bovine lymphocytes and monocytes 24-96 h post-exposure to the virus but there was no apparent replication of the virus in ovine alveolar macrophages during the culture period. The virus replicated to higher but statistically insignificant titres in ovine and bovine peripheral blood monocytes than in lymphocytes, with lymphocytes yielding peak titres significantly earlier. The secondary cell lines obtained from ovine liver and bone marrow also supported the replication of BRSV to high titres. The titres of BRSV in ovine and bovine lymphocytes and monocytes were significantly lower than in secondary cell lines. The addition of human recombinant tumour necrosis factor alpha after exposure to the virus or pre-incubation of ovine or bovine monocytic cells with either human recombinant interleukin 2 or phorbol myristate acetate before exposure to BRSV, did not significantly affect virus titre. Pre-incubation of cells with indomethacin or actinomycin significantly lowered virus titre (p < 0.05).

Serology of Orthopoxvirus cameli infection in dromedary camels: analysis by ELISA and western blotting.

An enzyme-linked immunosorbent assay (ELISA) was developed, together with a Western blotting technique, for the detection of total and IgG and IgM antibodies to camelpox virus (Orthopoxvirus cameli) in camel (Camelus dromedarius) sera and for identifying the seroreactive antigens of the virus. A total of 520 camels from different regions in Libya were tested. The overall seropositivity rate in the examined herds was 9.8%, and varied between herds from 0 to 30%. Two viral antigenic determinants (31 and 35 kDa) were shared by the Western blotting patterns of all the positive camel sera tested. The developed ELISA assay showed ability to differentiate between orthopox and parapoxvirus infections in camels. It is considered that the ELISA technique is justified for serodiagnosis of camelpox in the camel and could be easily modified and usefully applied to other species at risk of poxvirus infection.

The effects of bovine respiratory syncytial on normal ovine lymphocyte responses to mitogens or antigens in vitro.

In the present study peripheral blod mononuclear cells (MNC) obtained from normal uninfected lambs were used to study the possible effects of bovine respiratory syncytial virus (BRSV) on lymphocyte responses to the mitogens, phytohaemagglutinin (PHA), concanavalin A (Con A) and pokeweed mitogen (PWM) in vitro. Live BRSV had a depressive effect on the proliferative responses of normal MNC to PHA, Con A and PWM. Inactivated BRSV and a commercial preparation of prostaglandin E2 were also found to depress the proliferative responses of normal ovine MNC to PHA but recombinant tumour necrosis factor-alpha (TNF-alpha) had no such effect. Serum samples obtained from BRSV-infected lambs contained substances inhibitory to PHA-driven lymphocyte blastogenesis. Memory blastogenic responses to border disease virus (BDV) of lymyphocytes obtained from lambs previously primed with BDV were significantly reduced when lymphocytes were exposed to infectious BRSV.