Antigen-specific antibody isotypes, lymphocyte subsets and cytokine profiles in cattle naturally infested by Hypoderma sp. (Diptera: Oestridae).

Antigen-specific antibody responses, T cell subsets and cytokine profiles were studied in 7 heifers naturally infested by Hypoderma sp. in Northwestern Spain. Immunoglobulin G (IgG) levels increased significantly at the end of the endogenous life cycle of the parasite (Mr). Similarly, IgG1 subclass increased considerably when first instars (L1) started their migration towards the back (Nv-Ja), whereas IgG2 increased earlier, coinciding with the arrival of L1 to the resting sites (Jn-Jl). Both subclasses decreased significantly when L3 began to leave the host. IgM levels and CD4 and CD8 profiles hardly oscillated throughout the life cycle of the parasite into the host. The CD4/CD8 ratio showed helper T cell predominance. Serum interferon-γ (IFN-γ) concentrations decreased from October to the end of the study. Interleukin 4 (IL-4) concentrations decreased in January and increased in February and May. There were a significant positive relationship between IL-4 and IgG2 subclass and a negative correlation between IFN-γ, IgG and IgG1 and also between IgM and CD2 and CD8 counts. These results suggest that in the early phases of natural primoinfestations by Hypoderma there is a slight predominance of a Th1 response, characterized by high IgG2 and IFN-γ levels, which is followed by a Th2 response with a clear predominance of IgG1 and IL-4.

Prevalence of mixed trematode infections in an abattoir receiving cattle from northern Portugal and north-west Spain.

Between February 2007 and January 2009, a total of 776 slaughtered animals were examined for the presence of Trematoda in the liver, gall bladder, oesophagus and stomach (rumen and reticulum). Data collected were analysed by the age and breed of the animals. The percentage of cattle from which Trematoda were found was 38 per cent (95 per cent confidence interval [CI] 35 to 41 per cent); 28 per cent (95 per cent CI 25 to 31 per cent) had Fasciola hepatica, 12 per cent (95 per cent CI 10 to 14 per cent) Calicophoron daubneyi and 6 per cent (95 per cent CI 4 to 8 per cent) Dicrocoelium species. A significantly high prevalence of fasciolosis and dicrocoeliosis was observed in cattle over 10 years of age. Autochthonous Rubia Gallega cattle had the highest prevalence of fasciolosis and crossbred cattle had the highest prevalence of dicrocoeliosis (P<0.05). Twenty per cent (95 per cent CI 15 to 25 per cent) of the cattle positive for Fasciola also had Calicophoron species; 10 per cent (95 per cent CI 6 to 14 per cent) also had small liver flukes (Dicrocoelium species).

Immunohistochemical characterization of inflammatory cells in the skin of cattle undergoing repeated infestations with Hypoderma lineatum (Diptera: Oestridae) larvae.

Cutaneous T- and B-cell responses were studied in three groups of cattle (n=4) after a primary (G1), secondary (G2) and tertiary (G3) experimental infestation with Hypoderma lineatum first instars (L1). Cattle were each infested with 25 L1 deposited onto the skin. Skin biopsies were taken at 0, 6, 12, 48, 96 and 144 h post infestation (hpi). The kinetics of infiltration of CD3(+), CD4(+) and CD8(+) T lymphocytes and B cells at the infestation site was evaluated immunohistochemically. The number and pattern of infiltration of CD3(+) T cells were similar in the different groups of animals, showing a progressive increase until 96 hpi. The number of CD4(+) T helper cells increased significantly at 96, 6 and 48 hpi in G1, G2 and G3, respectively. CD8(+) cytotoxic T-cell infiltration was similar in the different groups, except at 48 hpi when there was a significant difference between G1 and G3. The CD4:CD8 ratio showed predominance of CD4(+) cells throughout the response. The number of B cells increased with the number of previous infestations, with significant differences between G1 and G3 at 12 hpi. All of the cell types concentrated around the superficial and deep dermal vessels. During primary larval infestations there was a predominance of CD4(+) cells, whereas in cattle sensitized by previous infestations, B cells were most abundant in the infiltrate. These findings suggest that humoral immunity may play a significant role in bovine immunity to H. lineatum.

Protostrongylid infection in meat sheep from Northwestern Spain: prevalence and risk factors.

2093 Faecal samples from 74 commercial meat ovine flocks were collected and examined by the Baermann-Wetzel method for protostrongylid infection. The risk of being infected by lungworms was evaluated with a data mining classification tree (CHAID), and the intensity of infection with a general linear model (GLM). 242 out of 2093 faecal samples examined were positive for protostrongylid infection (11.6%; 95% CI 10.2-12.9). Only two species were found, Muellerius capillaris (97.9%) and Neostrongylus linearis (5.4%). 50 out of 74 farms presented at least one animal shedding protostrongylid larvae in faeces. All of them held animals infected by M. capillaris and seven presented mixed infections with N. linearis. Average larval output in infected sheep was 11.9 (SD 30.91). This study showed that protostrongylid prevalence in sheep for meat production was determined mainly by a positive interaction with Dictyocaulus filaria infection; other factors that have influenced over protostrongylid infection were age, introducing external animals in the flocks, mixed management with goats and animal density in pastures. Treatment effects on prevalence were only observed in flocks that did not introduce ewes. The lowest protostrongylid prevalence has been reported in flocks without D. filaria infection and without contact with goats.

Efficacy of anthelminthic control programs against natural Muellerius capillaris infection in sheep in the north-west of Spain. Effect on blood gases and pH in venous blood samples.

The field efficacy of a single dose treatment against natural M. capillaris infection using different anthelmintic drugs, extensively employed in ovine parasite control programs in Galicia (N.W. Spain), and the effect of protostrongylid infection on ovine respiratory functions, were evaluated. Five groups (n = 5) of ewes were used in this study; animals were treated with injectable ivermectin 10.2 mg/kg), levamisole (7.5 mg/kg) and albendazole (5 mg/kg) and monitored at 0, 7, 21, 42 and 63 days posttreatment (d.p.t.) by enumeration of the lungworm larvae per gram of faeces (l.p.g.) and determination of gas tension and pH in venous blood using an i-Stat portable clinical analyzer. No statistical difference was found either in larval elimination between untreated and treated groups or in the reduction in larval counts in all of the treated groups. A significant increase in partial oxygen tension (pO2) and saturation (sO2) between day 0 and 7 p.t. was observed in all treated animals. These values decreased significantly at day 21 to previous levels. There were no statistical differences in blood gases between uninfected and treated groups. We can conclude that under Galician field conditions, parasitic control programs are not totally effective against M. capillaris infection.

Application of the analysis of serum antibodies (immunoglobulins M and G) to estimate the seroprevalence of ovine oestrosis and to evaluate the effect of chemotherapy.

The analysis of immunoglobulin (Ig) M and IgG serum isotypes as indicators of the presence of Oestrus ovis developmental stages was carried out. A serological survey to discover the seasonal variations in the prevalence of oestrosis in sheep from an oceanic climate area was developed. Six hundred and sixty-nine blood samples were analyzed for the presence of IgM and IgG using an enzyme-linked immunosorbent assay and O. ovis second-stage larvae excretory/secretory antigens. In addition, the effect of an ivermectin-based treatment on the humoral immune response was measured. The percentage of positive animals was 54% for IgM and 55% for IgG. The highest percentages of sheep positive to IgM were recorded in the winter and to IgG in the summer. No animals positive to IgM were detected in the ivermectin-treated sheep. These results seem to underline that oestrosis is a parasitic disease to keep watch for in regions with an oceanic climate. A noninvasive evaluation of the success of chemotherapy seems possible by analyzing the serum IgM response.

Seroprevalence of Toxoplasma gondii and Neospora caninum in wild and domestic ruminants sharing pastures in Galicia (Northwest Spain).

The prevalence of antibodies to the protozoan parasites Toxoplasma gondii and Neospora caninum were investigated by the direct agglutination test (DAT) and cELISA, respectively, in 160 roe deer (Capreolus capreolus), 177 sheep and 178 cattle sharing pastures in Galicia (Northwest Spain). The seroprevalence for T. gondii was 13.7% in roe deer, 57% in sheep and 7.3% in cattle. The seroprevalence for N. canimum was 6.8%, 10.1% and 24.1% in roe deer, sheep and cattle, respectively. Statistically significant differences were observed between sheep and the other species for T. gondii and between cattle and the other ruminants for N. caninum. Only 19/515 animals were positive for both, T. gondii and N. caninum. Statistically significant differences were observed among different geographical areas for T. gondii but not for Neospora, seroprevalence being higher in the coastal area lower than in other areas. This study reveals a widespread exposure to T. gondii in Galician ruminants, and therefore, those species, particularly sheep, should be regarded as a potential source of infection for humans.

Local and systemic cytokine responses during larval penetration in cattle experimentally infested with Hypoderma lineatum (Diptera: Oestridae).

Local and systemic cytokine responses were studied in 3 groups of cattle, with 4 animals each, experimentally infested with Hypoderma lineatum (De Villers) first instars (L1). The first group was undergoing a primary infestation (G-1), the second group was undergoing a secondary infestation (G-2) and the third group was infested for their third consecutive year (G-3). Cattle were infested with 25 L1 deposited on the skin. Blood and skin samples were taken at 0, 6, 12, 48, 96 and 144h post-infestation (h.p.i.). Interleukin 10 (IL-10), IL-4 and interferon gamma (IFN-gamma) production was studied by immunohistochemistry and sandwich ELISAs. IL-4(+) cells showed a significant increase at 6h.p.i. in both reinfested groups (G-2 and G-3) when compared with G-1. In all groups the number of IL-4(+) cells decreased significantly at 48h.p.i. IL-10(+) cells increased in G-1 at 6 and 48h.p.i., whereas in both reinfested groups increased at 12h.p.i. with a peak at 48h.p.i. IFN-gamma(+) cells showed a significant increment at 6h.p.i. in all groups, followed by a rapid descent at 12 (G-1 and G-2) and 48h.p.i. (G-3). Penetration of the skin by H. lineatum did not have any significant effect on IFN-gamma serum concentrations and, except for IL-10 there were no correlation between local production and serum concentrations of cytokines. The increase of both Th1 (IFN-gamma) and Th2-type cytokines (IL-4 and IL-10) indicates that bovine T-cell response during the first phases of the infestation by H. lineatum is apparently a Th0 response.

Immunomodulatory effect of Hypoderma lineatum antigens: in vitro effect on bovine lymphocyte proliferation and cytokine production.

This study examines the immunomodulatory effect of a crude larval extract (CLE), obtained from first stage larvae (L1) of H. lineatum, and the purified fractions hypodermin A (HyA), HyB and HyC. Proliferative responses of peripheral blood mononuclear cells (PBMC) from uninfested and previously infested cattle and the production of the cytokines IL-10, IL-4 and IFN-gamma, in response to concanavalin A (Con A), were determined. The stimulation index of peripheral blood mononuclear cells from uninfested cattle was significantly lower than that from infested animals with the different antigens assayed. The HyA was the antigen that most inhibited the proliferative response, followed by the HyB, the HyC and the CLE. This hypodermin provoked an increase of IFN-gamma and a suppression of IL-10 production that would support a Th1-like cytokine response. The HyB reduced the production of IL-10 stimulated by the Con A in cultures from infested animals. The HyC did not modulate the production of cytokines. Finally, the CLE induced a marked suppression in the production of the different cytokines in cultures from naïve and previously sensitized animals. Our results indicate that Hypoderma larval secretions are comprised of different components (hypodermins) that individually induce distinct but partially overlapping modulatory responses.

Intrinsic Factors Influencing the Infection by Helminth Parasites in Horses under an Oceanic Climate Area (NW Spain).

A coprological survey to determine the influence of some intrinsic factors (breed, age, and sex) on the infection by helminth parasites in equine livestock (n = 418) under an oceanic climate area (NW Spain) was conducted. Faecal samples were individually collected and analyzed by the coprological techniques. The main strongylid genera identified were Trichonema and Cyalocephalus spp (small strongyles) and Strongylus and Triodontophorus (large strongyles). The prevalence of gastrointestinal nematode was 89% (95% CI 86, 92) and 1% cestoda (0, 2). The percentage of horses with strongyloid parasites was 89% (86, 92), 11% (8, 14) for Parascaris, and 3% (1, 5) for Oxyuris. The highest prevalence for ascariosis was observed in the youngest horses (<3 years), for oxyurosis in the >10 years animals, and for strongylosis in the 3-10 years ones. Females were significantly more parasitized than males. A negative correlation between the age and the egg-excretion of ascarids and strongyles was recorded. The autochthonous and the English Pure Blood horses were the most parasitized. We concluded that the infections by helminths, especially the strongyloids, are significantly common in the region, so that greater importance should be given to this situation.

Occurrence and larval growth of Hypoderma lineatum in the oesophagi of cattle from northwest Spain: influence of geographical and climatic conditions.

From March 2004 to March 2005, 862 bovine oesophagi were collected at weekly intervals from a slaughterhouse in Lugo in northwest Spain in order to establish the occurrence and chronobiology of the first-instar (L1) oesophageal phase of Hypoderma lineatum (De Villers) (Diptera: Oestridae) in different climatic areas from this region. First instars were observed in oesophagi from May to February (10 months/year). The annual prevalence was 18.2%. Maximum prevalence occurred in July and October. Prevalence in the coastal area was significantly lower than in the other areas. A total of 846 L1s were extracted from 120 infested oesophagi, 814 from the submucosa and 32 from external locations. The mean intensity of infestation was low (7.6 +/- 6.2); only 3.3% of the oesophagi harboured more than 30 L1s. Maximum numbers of L1/animal were observed in November and January. There were no differences in intensity of infection between climatic areas. The length of L1s ranged from 4.6 +/- 0.86 mm in May to 14.6 +/- 1.04 mm in January. The growth of larvae collected from external sites was parallel to those found in the submucosa. Mountainous areas produced the L1s of greatest length, followed by central and transition zones and, finally, coastal zones. To avoid adverse reactions resulting from the application of systemic insecticides, it will be necessary to adjust the time at which cattle are treated according to geographical or climatic area.

Evaluation of an antigen capture ELISA for the early diagnosis of Hypoderma lineatum in cattle under field conditions.

An antigen capture or sandwich ELISA (sELISA) was evaluated for the diagnosis of Hypoderma lineatum in cattle under field conditions in northwestern Spain. The kinetics of circulating hypodermin C (HyC) and specific antibodies during the course of natural infestation were determined in a group of 10 Frisian calves. In addition, oesophagi and blood samples were taken from 105 cows at a slaughterhouse in order to compare three methods for the diagnosis of H. lineatum: sandwich ELISA for the detection of the antigen HyC (sELISA), indirect ELISA for the detection of antibodies anti-HyC (iELISA) and the detection of first instars (L1) in the oesophagus. In naturally infested cattle, HyC was present in circulation at low levels during the early and late phases of the infestation. However, in the middle phase, coinciding with the presence of L1 in the oesophagus, two peaks of increased HyC concentration were observed. Specific antibodies increased progressively until the first appearance of larvae in warbles on the back. There was no correlation between antigen or antibody levels and the number of grubs in the back. Prevalence of first instars in the oesophagi of slaughtered cows was 21.9% (23/105). The percentage of cattle that were positive for circulating antigen was slightly higher (24.8%), suggesting the recent destruction of migrating larvae in some animals. However, there was no correlation between the number of L1 and HyC levels. With the iELISA, 79% of the animals were positive to Hypoderma, which means that a high percentage of those animals have been exposed to the parasite but they had no apparent current infestation. The sELISA is a good tool to follow larval development within the host; however, the episodic elevation of HyC levels limits the usefulness of this test for the early diagnosis of Hypoderma under field conditions.