- Email: [email protected]
Antigen-specific antibody isotypes, lymphocyte subsets and cytokine profiles in cattle naturally infested by Hypoderma sp. (Diptera: Oestridae)
Veterinary Parasitology 184 (2012) 230–237
Contents lists available at SciVerse ScienceDirect
Veterinary Parasitology journal homepage: www.elsevier.com/locate/vetpar
Antigen-specific antibody isotypes, lymphocyte subsets and cytokine profiles in cattle naturally infested by Hypoderma sp. (Diptera: Oestridae) ˜ L. Vázquez, V. Dacal, C. López, P. Díaz, P. Morrondo, P. Díez-Banos, R. Panadero ∗ Departamento de Patología Animal: Sanidad Animal, Facultad de Veterinaria, Universidad de Santiago de Compostela, 27071 Lugo, Spain
a r t i c l e
i n f o
Article history: Received 24 March 2011 Received in revised form 12 August 2011 Accepted 5 September 2011 Keywords: Cattle-arthropoda Hypoderma Antibody isotype Cytokine Lymphocyte subsets
a b s t r a c t 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-␥ (IFN-␥) 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-␥, 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-␥ levels, which is followed by a Th2 response with a clear predominance of IgG1 and IL-4. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Hypoderma sp. (Diptera: Oestridae) larvae cause a subcutaneous myiasis mainly in grazing cattle. First stage larvae (L1) penetrate into the skin and migrate through connective tissues to reach the oesophageal submucosa (H. lineatum) or the epidural fat (H. bovis), where they overwinter for some months before migrating to sub-dermal tissues of the back. Second and third instar development takes place within subcutaneous granulomas (warbles). Mature third instars (L3) exit the host and pupate within a short period of time. Larvae migrating within the host and in
∗ Corresponding author. Tel.: +34 982822125; fax: +34 982822001. E-mail address: [email protected] (R. Panadero). 0304-4017/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2011.09.013
sub-dermal warbles provoke production losses and increased susceptibility to diseases (Drummond, 1987). Both cattle species (Hypoderma bovis, L. and Hypoderma lineatum, De Villers) coexist in Spain, but H. lineatum is observed much more frequently than H. bovis, whose populations are very scarce (Panadero et al., 1998; Reina et al., 1998). In Northwestern Spain, two main types of husbandry are practiced; one aimed at the production of meat, using Rubia Gallega and crossbreeds, where cattle is maintained on pasture in an extensive or semi-extensive system, favoring their contact with the parasite, and other at the production of milk, using Frisians and Brown Swiss that are maintained in an intensive system. Hypoderma sp. biology, as occurs with most of insects, is very much conditioned by weather conditions; climate
L. Vázquez et al. / Veterinary Parasitology 184 (2012) 230–237
directly influences the development of free stages of the parasite, flies and pupae, affecting the chronology of this myiasis and the intensity of infestation. In North-western Spain the period of fly activity of H. lineatum, usually starts in March–April, first instars (L1) can be found in the oesophageal submucosa from June to February and the appearance of warbles in the back (warble season) begins in January–February (Panadero, 1996; Panadero et al., 2007). First stage larvae of Hypoderma penetrate intact skin aided by enzymatic secretions (hypodermins) stored in their midgut (Nelson and Weintraub, 1972) and undertake a long migration through the deep connective tissue of their host, where they are continuously exposed to immune effector mechanisms. Infestation of cattle with Hypoderma sp. has been shown to induce an immunological response characterized by an increase in serum immunoglobulin G levels with a peak when the first warbles become detectable on the back and drop once L3 have emerged (Sinclair and Wassall, 1983; Boulard, 1985; Pruett and Barrett, 1985; Panadero et al., 1997, 2002). However, there is not apparent correlation between antibody titres and resistance to Hypoderma infestation in cattle (Pruett et al., 1989; Panadero, 1996). Although detailed analyses have been performed on humoral responses involving total IgG responses to Hypoderma there is no information available with regard to the production of different immunoglobulin isotypes and subclasses. Cattle develop acquired resistance after repeated exposures to larval antigens (Gingrich, 1982). This resistance is recognized as an important factor in controlling Hypoderma populations and depends upon the number of previous exposures and the number of larvae infesting the host (Baron and Weintraub, 1987). Numerous studies have implicated the isotype of antibodies in the effective protection against diseases (Brown et al., 1999; Avramidis et al., 2002; Hagiwara et al., 2005; Almería et al., 2009; Piper et al., 2009). IgG subclasses in both the murine and human systems have been reported to be differentially regulated by cytokines, which act as switch and growth factors for B cells at different stages of maturation (Stavnezer, 1996). Cytokine control of antibody production in cattle may differ from the regulatory pathways, which have been described in the mouse and human (Estes, 1996). It is clear from studies on the T lymphocyte response that the Th1/Th2 paradigm, postulated by Mosmann et al. (1986) in a murine model, is less well defined in ruminants (Estes and Brown, 2002). It is also well established that Th1 and Th2 cytokines have an influence on the antibody production and isotype expression; for instance, recombinant bovine IL-4 up regulates the expression of IgM, IgG1 and IgE in vitro (Estes, 1996). Cytokine responses and lymphocyte subsets during larval penetration in cattle experimentally infested by H. lineatum have been determined in recent studies (Dacal et al., 2009, 2011). At this early phase of the infestation there is an increase of both Th1 (IFN-␥) and Th2-type cytokines (IL-4 and IL-10) suggesting a Th0 response. However, there is no information describing the subsequent phases of larval migration, especially during their residence in the oesophageal submucosa and after their arrival at the subcutaneous tissue of the back.
231
The aim of this study was to study the dynamics of antigen-specific antibodies (IgM, IgG, IgG1 and IgG2), T lymphocyte subsets (CD2, CD4 and CD8) and cytokine profiles (IFN-␥ and IL-4), and their possible relationships during the course of natural infestations by Hypoderma in cattle. 2. Materials and methods 2.1. Animals and blood samplings The trial was carried out on a beef farm with a history of hypodermosis. Identification of third instars (L3) obtained in previous infestations on the farm, showed the ˜ presence of H. lineatum. On this farm, located in A Coruna (Northwestern Spain), cows are raised in a semiextensive grazing system, allowing natural infestations by Hypoderma sp., whereas calves are maintained indoors until weaning (8–11 months of age) them they are destined for the slaughterhouse. They fed milk from their mothers and concentrates are gradually introduced in their diet. The herd received benzimidazole treatments twice a year, but drugs for lice or Hypoderma control were not used routinely and no fly control product was used during the summer months. Routine faecal examinations, using flotation and sedimentation techniques were negative to parasitic forms. Seven 1-year-old Rubia Gallega heifers were selected for this study on the basis of the presence of both anti-Hypoderma antibodies and the parasite antigen hypodermin C (HyC), as determined by an indirect (Panadero et al., 1997) and sandwich ELISA (Panadero et al., 2002), respectively. Those animals were in their 1st grazing year, thus it was considered that they were undergoing their first infestation by Hypoderma. All the animals were bled monthly (May 2007–May 2008) by caudal venipuncture, covering the entire endogenous cycle of H. lineatum in our region (Panadero, 1996; Panadero et al., 2007). Warbles were counted by manual palpation at monthly intervals. Results are expressed as the mean number of warbles per infested animal. Infested animals were treated subcutaneously with ivermectin (Ivertin® injectable, Laboratorios Calier, Spain) at the recommended dose (0.2 mg/kg p.v.) 2 months before the end of the study (March 2008). An uninfested control group, comprised by four Rubia Gallega calves, was also bled at monthly intervals. Control calves were born at the farm and were maintained indoors, avoiding their contact with the parasite. They were 3month-old at the beginning of the study and they remained at the farm until weaning (11 months of age). In consequence, control calves were only available for the first 8 months of the trial and their results are expressed as mean (SD) of the individual values obtained from May to December 2007. 2.2. ELISA protocols 2.2.1. Antibody detection Serum samples were processed by an indirect ELISA test described by Panadero et al. (1997) for IgG detection against the antigenic fraction hipodermin C (HyC).
L. Vázquez et al. / Veterinary Parasitology 184 (2012) 230–237
Abs (490 nm)
a
1,4
14
1,2
12
1,0
10
0,8
8
0,6
6
0,4
4
0,2
2
0,0
Warbles/infested animal
232
0 My
Jn
Jl
Au
St
Oc
Nv
Dc
Ja
Fb
Mr
Ap
My
Month
Abs (490 nm)
b
Control
1,4
14
1,2
12
1,0
10
0,8
8
0,6
6
0,4
4
0,2
2
0,0
Warbles/infested animal
IgG
0 My
Jn
Jl
Au
St
Oc
Nv
Dc
Ja
Fb
Mr
Ap
My
Month IgM
Control
Fig. 1. Kinetic of the antigen-specific IgG (a) and IgM (b) responses in cattle during natural infestations by Hypoderma sp. Bars represent the intensity of infestation. Vertical lines indicate standard deviation and horizontal line represent mean value of controls.
Horseradish peroxidase-conjugated sheep anti-bovineIgG1 (ShAB IgG1-HRP, 1/5000), ShAB IgG2 (1/500) and ShAB IgM (1/20000) were purchased from Serotec (Oxford, UK). Positive and negative control sera were introduced in every plate to normalize absorbance values. 2.2.2. Cytokine detection IL-4 and IFN-␥ serum levels were determined by sandwich ELISAs that specifically detect soluble cytokine proteins according to the protocols described in Dacal et al. (2009). Serum concentrations (pg/ml) were calculated according quadratic curves using recombinant bovine IL-4 and IFN-␥ (Serotec, Oxford, UK). 2.3. Flow cytometric analysis of lymphocyte subsets Lymphocytes were analyzed for expression of cellsurface differentiation antigens by direct immunofluorescence labelling. Briefly, fresh blood collected into sterile acid citric dextrose (ACD) tubes was incubated with fluorescein (FITC)-conjugated monoclonal antibodies against bovine CD2, CD4 and CD8 (Serotec, Oxford, UK) or an isotype control (Mouse IgG1 and IgG2a: FITC) at room temperature for 30 min. Then 2 ml of PBS/BSA 1% was added to each tube and were centrifuged at 500 × g for 10 min and the supernatant was discarded. Red cells were lysed
with 2 ml of lysis buffer BD FACSTM Lysing Solution (Beckton Dickinson). The mix was incubated at 25 ◦ C in agitation for 10 min and then centrifuged at 500 × g for 5 min and the supernatant discarded. Finally, the cells were resuspended with 400 l de PBS/BSA 1%. Labelled cells were detected in an Epics XL (Coulter Corporation, USA) cytometer. Data from 50,000 cells per sample were acquired using a 15 mW air-cooled argon ion laser with excitation wavelength of 488 nm. The results were analyzed with the System II program (Coulter Corporation, USA). Data are expressed as a percentage of positive cells. 2.4. Statistics Data were analyzed using the Statistical Package for Social Sciences (SPSS), Version 18.0.1 (SPSS Inc., Chicago, IL). Differences in the different parameters over time were studied by repeated measures ANOVA using the repeated contrast as a within-subject contrast to compare adjacent measures (P < 0.05). One sample t-test was used to determine whether the average values of the different parameters from control calves differed significantly from monthly values of primoinfested cows. Correlations between specific antibodies, lymphocyte subsets and cytokine concentrations were assessed using the Spearman rank correlation test.
Abs (490 nm)
a
233
1,4
14
1,2
12
1,0
10
0,8
8
0,6
6
0,4
4
0,2
2
Warbles/infested animal
L. Vázquez et al. / Veterinary Parasitology 184 (2012) 230–237
0
0,0
My
Jn
Jl
Au
St
Oc
Nv
Dc
Ja
Fb
Mr
Ap
My
Month
Abs (490 nm)
b
Control
1,4
14
1,2
12
1,0
10
0,8
8
0,6
6
0,4
4
0,2
2
0,0
Warbles/infested animal
IgG1
0 My
Jn
Jl
Au
St
Oc
Nv
Dc
Ja
Fb
Mr
Ap
My
Month IgG2
Control
Fig. 2. Kinetics of the subclasses IgG1 (a) and IgG2 (b) in cattle during natural infestations by Hypoderma sp. Bars represent the intensity of infestation. Vertical lines indicate standard deviation and horizontal line represent mean value of controls.
3. Results 3.1. Warble counts Warble appearance and the intensity of infestation are represented in Figs. 1–4. Four out of seven heifers presented cattle warbles with a mean of 11.2 (1.33 SD) warbles per infested animal (range 1–30 warbles). Warbles appeared in February, reaching a peak in this month. One month later the number of warbles had considerably decreased (1.3; 0.57 SD) and no nodules were observed in April, after the administration of a treatment. 3.2. Antigen-specific antibody responses IgG levels at the early stages of the infestation were relatively elevated (Fig. 1a), but they increased considerably once L1 arrived to the back, with a significant increment in March and a subsequent descent in May. Mean OD value for control calves was 0.31 (0.149 SD). One sample t-test showed significant differences between fixed mean IgG values from control group and primoinfested cattle from June to December 2007 and from February to April 2008. IgM maintained constant levels throughout the study (Fig. 1b). Mean OD value for control calves (0.11; 0.046 SD)
was significantly lower to that of infested cattle along the study. The evolution of the subclasses IgG1 and IgG2 are represented in Fig. 2. IgG1 profiles at the beginning of the infestation remained relatively constant, and they increased significantly in November, December and January. Once warbles appeared on the back, IgG1 levels started to decline. Infested animals showed higher OD values (P < 0.05) than the mean of controls (0.18; 0.162 SD) in July, August and from December to April. IgG2 profiles increased significantly in June, July and October, and decreased in November. Mean OD in controls for IgG2 (0.06; 0.025 SD) was significantly higher than in those infested in May, June, September, December and April. 3.3. Changes in circulating lymphocyte subsets Fig. 3 shows the evolution of circulating lymphocyte subsets. The percentage of CD2+ cells was relatively elevated at the beginning of the study (36.7%), and showed a significant increment in January, with a peak in February, coinciding with the apparition of warbles on the back. Control animals showed a mean percentage of CD2+ cells of 21.9% (5.17 SD); this value was significantly lower than those of the infested animals in May and October 2007.
234
L. Vázquez et al. / Veterinary Parasitology 184 (2012) 230–237 14
100
12
80
10 60
8 6
40
4 20
Warbles/infested animal
% Lymphocytes
a
2
0
0 My
Jn
Jl
Au
St
Oc
Nv
Dc
Ja
Fb
Mr
Ap
My
Month CD2
100
14 12
80
10 60
8 6
40
4 20
Warbles/infested animal
% Lymphocytes
b
Control
2
0
0 My
Jn
Jl
Au
St
Oc
Nv
Dc
Ja
Fb
Mr
Ap
My
Month CD4
100
14 12
80
10 60
8 6
40
4 20
Warbles/infested animal
% Lymphocytes
c
Control
2
0
0 My
Jn
Jl
Au
St
Oc
Nv
Dc
Ja
Fb
Mr
Ap
My
Month CD8
Control
Fig. 3. Systemic lymphocyte subpopulations, CD2 (a), CD4 (b), CD8 (c) in blood of cattle naturally infested by Hypoderma determined by flow cytometry. Bars represent the intensity of infestation. Vertical lines indicate standard deviation and horizontal line represents mean value of controls.
CD4 and CD8 percentages did not varied significantly along the study. Mean percentage for control calves was 10.9 (2.01 SD) and 7.3 (1.91 SD) for CD4 and CD8, respectively. Mean CD4 values did not differ from those of the infested animals along the trial, and for CD8 differences were only significant in May 2007. CD4/CD8 ratios were always higher than 1 throughout the infestation, with the highest ratio in October (1.8).
3.4. Evolution of circulating cytokines The evolution of serum concentrations of the cytokines IFN-␥ and IL-4 are represented in Fig. 4. IFN-␥ showed two significant drops in October and January. The concentration of this cytokine in control animals was 11.3 pg/ml (8.54 SD), and this value differed significantly from the concentration found in the infested group in January and May 2008.
L. Vázquez et al. / Veterinary Parasitology 184 (2012) 230–237
100
14 12
80
10 60
8 6
40
4 20
2
0
Warbles/infested animal
Concentration (pg/ml)
a
235
0 My
Jn
Jl
Au
St
Oc
Nv
Dc
Ja
Fb
Mr
Ap
My
Month IFN-γ
100
14 12
80
10 60
8 6
40
4 20
2
0
Warbles/infested animal
Concentration (pg/ml)
b
Control
0 My
Jn
Jl
Au
St
Oc
Nv
Dc
Ja
Fb
Mr
Ap
My
Month IL-4
Control
Fig. 4. Serum concentrations of IFN-␥ (a) and IL-4 (b) in pg/ml in cattle naturally infested by Hypoderma. Bars represent the intensity of infestation. Vertical lines indicate standard deviation and horizontal line represents mean value of controls.
Circulating IL-4 levels showed a significant decrease in January followed by an increase in February and May. Mean concentration for control animals was higher than that of the infested group in January and lower in May 2008. 3.5. Correlations between specific antibodies, lymphocyte subsets and cytokine concentrations IgM antibodies showed a strong negative correlation with CD2 ( = −0.358; P = 0.009) and with CD8 counts ( = −0.340; P = 0.014). IFN-␥ concentrations were negatively correlated with the IgG isotype ( = −0.218; P = 0.038) and with the IgG1 subclass ( = −0.219; P = 0.037), whereas IL-4 values were positively correlated with IgG2 subclass ( = −0.217; P = 0.038). 4. Discussion This study investigates the isotype pattern, lymphocyte subsets and cytokine profile during the course of natural infestations by Hypoderma as a contribution to increase the understanding of the pathways of stimulation of the immune system in this myiasis. Traditionally, antibody titres are not considered a reliable guide to the level of resistance against Hypo-
derma (Pruett et al., 1989; Panadero, 1996); however, their involvement in protection mechanisms has not been excluded and some vaccine studies aiming at inducing high levels of antibodies have been very promising (Pruett et al., 1987; Colwell, 2011). In our work, specific IgG antibody levels were similar to those reported in several other studies (Sinclair et al., 1984; Colwell and Baron, 1990; Panadero et al., 1997), with maximum levels coinciding with the presence of warbles on the back. The IgG1 subtype predominated over IgG2 during the entire life-cycle of the parasite, except in October (IgG1/IgG2 ratio 0.648). The maximum IgG1/IgG2 ratio was detected in January (3.8). IgG2 subclass showed an early elevation, which would coincide with the arrival of H. lineatum L1 to the oesophagus. Both subclasses decreased significantly when L3 began to leave the host. Our results suggest that Hypoderma infestation provokes an intense humoral response, characterized by an initial elevation of IgG2, which would be followed by a phase with a clear predominance of specific IgG1. In contrast, specific IgM remained constant throughout the study, although with higher levels than controls. According to Gold and Defranco (1994), this type of response would indicate a T-dependent antibody response, characterized by an extensive switching to immunoglobulin isotypes other than IgM; whereas an antibody response to T-independent antigens would be dominated by IgM. Despite the relatively
236
L. Vázquez et al. / Veterinary Parasitology 184 (2012) 230–237
weak IgM response, there was a negative correlation with CD2 and CD8 subsets. Nevertheless, the mechanisms of this relation remain to be elucidated. In our study CD2+ cells remained at low levels in the early stages of the infestation and showed a significant increase in the later phases of the endogenous cycle of the parasite. Similarly, López et al. (2005) observed a low lymphocytic perivascular infiltration in primary infested animals in the early stages of the infestation. In a previous study, Dacal et al. (2009) reported a local predominance of helper over cytotoxic T cells during the phase of larval penetration. In this study, we have confirmed that this predominance persists along the entire life-cycle of the parasite into the host. According to several authors (Broadmeadow, 1984; Bowles et al., 1992, 1994; Colditz et al., 1996), T CD4+ cells are also implicated in the immune response against Lucilia cuprina in sheep, whereas T CD8 cells are scarce in primary infestations. In murine systems, IL-4 and IFN-␥ reciprocally regulate isotype production directly via their actions on B cells and indirectly through T helper cell differentiation pathways leading to Th1 or Th2 responses (see review by Estes, 1996). Regarding the kinetics of IFN-␥, serum concentrations remained relatively constant during larval penetration and migration towards the oesophagus and they began to decline in October; this decrease continued until the end of the endogenous parasite life-cycle. Similarly, Dacal et al. (2009) did not observe significant variations of this cytokine during larval penetration in cattle experimentally infested by H. lineatum. On the contrary, Bowles et al. (1994) reported increasing levels of IFN-␥ after the infestation by L. cuprina. Serum concentrations of IL-4, a key regulator of humoral and acquired immune responses, did not show significant variations until the last phases of larval development. Thus, there was a significant but transient decline just before the arrival of L1 to subcutaneous tissues and an increment once warbles appeared on the back. Our results revealed a negative correlation between IFN-␥ serum concentrations and IgG and IgG1 antibodies, but surprisingly we did not find any relationship with IgG2, which has been reported to be up regulated by this cytokine (Snapper and Paul, 1987). Therefore, the negative correlation between IgG and IFN-␥ is mainly due to IgG1. In contrast with Estes (1996), who demonstrate that recombinant bovine IL-4 up regulated the expression of IgG1 in vitro, we found a positive correlation between the IL-4 and the IgG2 subclass. Once analyzed the evolution of the different parameters together it can be concluded that, as stated by Estes and Brown (2002), the polarisation of the immune response is a dynamic process with variations throughout the different phases of development of the parasite into the host. In our study we have observed that, in the early stages of the infestation (larval penetration and arrival to the oesophagus), there is a slight predominance of a Th1 response characterized by relatively elevated IgG2 and IFN-␥ levels. The dominance of this type of response would result in a stimulation of cellular responses against the parasite which may be effective in killing some of the entering larvae, resulting in a degree of resistance. In our study, only four out of seven
infested animals were palpation positive, although the intensity of infestation was elevated in comparison with that of reinfested cows (data not shown). Panadero et al. (2009) found that larval antigens are capable to combine the activation of cellular responses with an immunosuppressive effect, especially in naïve cattle. However, further studies are necessary to study the mechanisms implicated in the development of acquired resistance after successive reinfestations. Later, during the mobilisation of L1 from the oesophagus and their arrival to the back, the immune reaction is more intense with a clear predominance of a Th2 response, characterized by high levels of IgG1 and IL-4. According to Gingrich (1980), most larvae that die in resistant hosts may not be able to reach the oesophagus, i.e. they would die shortly after entering their host. Thus, the primary effectors of acquired resistance are functioning in the skin and/or the connective tissues along the migration route and non-existent or non-functional in the oesophagus and subsequent portions of the migration route that lead to larval encapsulation in the back. Acknowledgements We are very grateful to the Rubia Gallega breeder “Pepe ˜ for his colde Miralles” (Sobrado dos Monxes, A Coruna) laboration in the realization of this study. This research was supported by the Research projects AGL-2004-01827 and AGL-2009-08939. Authors also thank the Spanish Ministry of Education and Science for L. Vázquez and V. Dacal pre-doctoral FPU grants. References Almería, S., Nogareda, C., Santolaria, P., Garcia-Ispierto, I., Yániz, J.L., López-Gatius, F., 2009. Specific anti-Neospora caninum IgG1 and IgG2 antibody responses during gestation in naturally infected cattle and their relationship with gamma interferon production. Vet. Immunol. Immunopathol. 130, 35–42. Avramidis, N., Victoratos, P., Yiangou, M., Hadjipetrou-Kourounakis, L., 2002. Adjuvant regulation of cytokine profile and antibody isotype of immune responses to Mycoplasma agalactiae. Vet. Microbiol. 88, 325–338. Baron, R.W., Weintraub, J., 1987. Lymphocyte responsiveness in cattle previously infested and uninfested with Hypoderma lineatum (De Vill.) and Hypoderma bovis (L.) (Diptera: Oestridae). Vet. Parasitol. 21, 43–50. Boulard, C., 1985. Avantages de l’immunodiagnostic de l’hypodermose bovine établi par hemaglutination passive et par ELISA à partir du sérum et du lactosérum sur la numeration des varrons. Ann. Rech. Vet. 16, 335–343. Bowles, V.M., Grey, S.T., Brandon, M.R., 1992. Cellular immune responses in the skin of sheep infected with larvae of Lucilia cuprina, the sheep blowfly. Vet. Parasitol. 44, 151–162. Bowles, V.M., Meeusen, E.N., Chandler, K., Verhagen, A., Nash, A.D., Brandon, M.R., 1994. The immune response of sheep infected with larvae of the sheep blowfly Lucilia cuprina monitored via efferent lymph. Vet. Immunol. Immunopathol. 40, 341–352. Broadmeadow, M., 1984. Pathogenesis of blowfly strike in sheep. Wool Technol. Sheep Breed. 32, 28–32. Brown, W.C., McElwain, T.F., Palmer, G.H., Chantler, S.E., Estes, D.M., 1999. Bovine CD4+ T-lymphocyte clones specific for rhoptry-associated protein 1 of Babesia bigemina stimulate enhanced immunoglobulin G1 (IgG1) and IgG2 synthesis. Infect. Immun., 155–164. Colditz, I.G., Eisemann, C.H., Tellam, R.L., Mcclure, S.J., Mortimer, S.I., Husband, A.J., 1996. Growth of Lucilia cuprina larvae following treatment of sheep divergently selected for fleece rot and fly strike with monoclonal antibodies to T lymphocyte subsets and interferon gamma. Int. J. Parasitol. 26, 775–782.
L. Vázquez et al. / Veterinary Parasitology 184 (2012) 230–237 Colwell, D.D., Baron, R.W., 1990. Early detection of cattle grub infestation (Hypoderma lineatum DeVill. and H. bovis L.) (Diptera: Oestridae) using ELISA. Med. Vet. Entomol. 4, 35–42. Colwell, D.D., 2011. Hidden antigens from third instar Hypoderma lineatum: impact of immunization on larval survival in artificial infestations. Vet. Parasitol. 175, 313–319. Dacal, V., Colwell, D.D., López, C., Pérez, V., Vázquez, L., Cienfuegos, S., Díaz, ˜ P., Morrondo, P., Díez-Banos, P., Panadero, R., 2009. Local and systemic cytokine responses during larval penetration in cattle experimentally infested with Hypoderma lineatum (Diptera: Oestridae). Vet. Immunol. Immunopathol. 131, 59–64. Dacal, V., López, C., Colwell, D.D., Vázquez, L., Díaz, P., Morrondo, P., Díez, P., Panadero, R., 2011. Immunohistochemical characterization of inflammatory cells in the skin of cattle undergoing repeated infestations with Hypoderma lineatum (Diptera: Oestridae) larvae. J. Comp. Pathol. 145, 282–288. Drummond, R.O., 1987. Economic aspects of ectoparasites of cattle in North America. In: Leanning, H.D., Guerrero, J. (Eds.), The Economic Impacts of Parasitism in Cattle. Proceedings of the MSD AGVET Symposium, 23rd World Veterinary Congress. Montreal, pp. 9–24. Estes, D.M., 1996. Differentiation of B cells in the bovine. Role of cytokines in immunoglobulin isotype expression. Vet. Immunol. Immunopathol. 54, 61–67. Estes, D.M., Brown, W.C., 2002. Type 1 and type 2 responses in regulation of Ig isotype expression in cattle. Vet. Immunol. Immunopathol. 90, 1–10. Gingrich, R.E., 1980. Differentiation of resistance in cattle to larval Hypoderma lineatum. Vet. Parasitol. 7, 243–254. Gingrich, R.E., 1982. Acquired resistance to Hypoderma lineatum: comparative immune response of resistant and susceptible cattle. Vet. Parasitol. 9, 233–242. Gold, M.R., Defranco, A.L., 1994. Biochemistry of B lymphocyte activation. Adv. Immunol. 55, 221–295. Hagiwara, K., Tokuda, M., Baba, T., Yamanaka, H., Kirisawa, R., Tsuji, M., Ishihara, C., Iwai, H., 2005. The role of IFN-␥ in cattle infected with Theileria sergenti. Vet. Parasitol. 127, 105–110. López, C., Colwell, D.D., Panadero, R., Paz, A., Pérez, J., Morrondo, P., Díez, P., Cascallana, J.L., Santamaría, V., Bravo, A., 2005. Skin immune responses in cattle after primary and secondary infections with Hypoderma lineatum (Diptera: Oestridae) larvae. Vet. Immunol. Immunopathol. 108, 285–294. Mosmann, T.R., Cherwinski, H., Bond, M.W., Giedlin, M.A., Coffman, R.L., 1986. Two types of murine helper T cell clone. I. Definition according to profiles of lymphokine activities and secreted proteins. J. Immunol. 136, 2348–2357. Nelson, W.A., Weintraub, J., 1972. Hypoderma lineatum (De Vill.) (Diptera: Oestridae): invasion of the bovine skin by newly hatched larvae. J. Parasitol. 58 (3), 614–624. Panadero, R., 1996. Estudio epidemiológico y de la respuesta inmune rrente a antígeno de Hypoderma lineatum como aportación a la lucha
237
contra la hipodermosis bovina en Galicia. Tesis Doctoral, Facultad de Veterinaria, Universidad de Santiago de Compostela, 255 pp. ˜ P., 1997. Effect Panadero, R., López, C., Mezo, M., Morrondo, P., Díez-Banos, of early treatment with ivermectin and doramectin on the dynamics of antibody response in cattle naturally infested by Hypoderma lineatum and H. bovis. Vet. Parasitol. 73, 325–334. Panadero, R., López, C., Paz, A., Díez, P., Morrondo, P., 1998. Estudio sobre el desarrollo de las larvas esofágicas de Hypoderma lineatum y su relación con el tratamiento precoz de la hipodermosis bovina en el Norte de ˜ Vet. México 29, 155–160. Espana. ˜ P., Colwell, Panadero, R., López, C., Parra, F., Morrondo, P., Díez-Banos, D.D., 2002. Detection of circulating hypodermin C: an antigen capture ELISA for diagnosis of cattle grub (Diptera: Oestridae) infestations. Vet. Parasitol. 108, 85–94. Panadero, R., Fernández, M., Vázquez, L., López, C., Dacal, V., Cienfuegos, ˜ P., 2007. Occurrence and larval S., Diaz, P., Morrondo, P., Díez-Banos, growth of Hypoderma lineatum in the oesophagi in cattle from Northwest Spain: influence of geographical and climatic conditions. Med. Vet. Entomol. 21, 225–230. Panadero, R., Dacal, V., López, C., Vázquez, L., Cienfuegos, S., Díaz, P., ˜ P., 2009. Immunomodulatory effect of Morrondo, P., Díez-Banos, Hypoderma lineatum antigens: in vitro effect on bovine lymphocyte proliferation and cytokine production. Parasite Immunol. 31, 72–77. Piper, E.K., Jonsson, N.N., Gondro, C., Lew-Tabor, A.E., Moolhuijzen, P., Vance, M.E., Jackson, L.A., 2009. Immunological profiles of Bos taurus and Bos indicus cattle infested with the Cattle Tick, Rhipicephalus (Boophilus) microplus. Clin. Vac. Immunol, 1074–1086. Pruett, J.H., Barrett, C.C., 1985. Kinetic development of humoral antiHypoderma lineatum antibody activity in the serum of vaccinated and infested cattle. Southwest. Entomol. 10, 39–48. Pruett, J.H., Barrett, C.C., Fisher, W.F., 1987. Kinetics development of serum antibody to purified Hypoderma lineatum proteins in vaccinated and non-vaccinated cattle. Southwest. Entomol. 12, 79–88. Pruett, J.H., Fisher, W.f., Temeyer, K.B., 1989. Evaluation of purified proteins of Hypoderma lineatum as candidate immunogens for a vaccine against bovine hypodermiasis. Southwest. Entomol. 14, 363–374. Reina, D., Martínez-Moreno, F., Hernández, S., Navarrete, I., 1998. Chronobiology of bovine hypodermosis in Europe. In: Boulard, C., Sol, J.; Pithan, K., O’Brien, D., Webster, K., Sampimon, C. (Ed.), Improvements in the Control Methods for Warble Fly in Livestock. Brussels, European Commission, p. 56. Sinclair, I.J., Wassall, D.A., 1983. Enzyme-linked immunosorbent assay for the detection of antibodies to Hypoderma bovis in cattle. Res. Vet. Sci. 34, 251–260. Sinclair, I.J., Tarry, D.W., Wassall, D.A., 1984. Persistence of antibody in calves after an infection with Hypoderma bovis. Res. Vet. Sci. 37, 383–384. Snapper, C.M., Paul, W.E., 1987. Interferon-gamma and B cell stimulatory factor-1 reciprocally regulate Ig isotype production. Science 236, 944–947. Stavnezer, J., 1996. Antibody class switching. Adv. Immunol. 61, 79–146.
Contents lists available at SciVerse ScienceDirect
Veterinary Parasitology journal homepage: www.elsevier.com/locate/vetpar
Antigen-specific antibody isotypes, lymphocyte subsets and cytokine profiles in cattle naturally infested by Hypoderma sp. (Diptera: Oestridae) ˜ L. Vázquez, V. Dacal, C. López, P. Díaz, P. Morrondo, P. Díez-Banos, R. Panadero ∗ Departamento de Patología Animal: Sanidad Animal, Facultad de Veterinaria, Universidad de Santiago de Compostela, 27071 Lugo, Spain
a r t i c l e
i n f o
Article history: Received 24 March 2011 Received in revised form 12 August 2011 Accepted 5 September 2011 Keywords: Cattle-arthropoda Hypoderma Antibody isotype Cytokine Lymphocyte subsets
a b s t r a c t 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-␥ (IFN-␥) 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-␥, 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-␥ levels, which is followed by a Th2 response with a clear predominance of IgG1 and IL-4. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Hypoderma sp. (Diptera: Oestridae) larvae cause a subcutaneous myiasis mainly in grazing cattle. First stage larvae (L1) penetrate into the skin and migrate through connective tissues to reach the oesophageal submucosa (H. lineatum) or the epidural fat (H. bovis), where they overwinter for some months before migrating to sub-dermal tissues of the back. Second and third instar development takes place within subcutaneous granulomas (warbles). Mature third instars (L3) exit the host and pupate within a short period of time. Larvae migrating within the host and in
∗ Corresponding author. Tel.: +34 982822125; fax: +34 982822001. E-mail address: [email protected] (R. Panadero). 0304-4017/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2011.09.013
sub-dermal warbles provoke production losses and increased susceptibility to diseases (Drummond, 1987). Both cattle species (Hypoderma bovis, L. and Hypoderma lineatum, De Villers) coexist in Spain, but H. lineatum is observed much more frequently than H. bovis, whose populations are very scarce (Panadero et al., 1998; Reina et al., 1998). In Northwestern Spain, two main types of husbandry are practiced; one aimed at the production of meat, using Rubia Gallega and crossbreeds, where cattle is maintained on pasture in an extensive or semi-extensive system, favoring their contact with the parasite, and other at the production of milk, using Frisians and Brown Swiss that are maintained in an intensive system. Hypoderma sp. biology, as occurs with most of insects, is very much conditioned by weather conditions; climate
L. Vázquez et al. / Veterinary Parasitology 184 (2012) 230–237
directly influences the development of free stages of the parasite, flies and pupae, affecting the chronology of this myiasis and the intensity of infestation. In North-western Spain the period of fly activity of H. lineatum, usually starts in March–April, first instars (L1) can be found in the oesophageal submucosa from June to February and the appearance of warbles in the back (warble season) begins in January–February (Panadero, 1996; Panadero et al., 2007). First stage larvae of Hypoderma penetrate intact skin aided by enzymatic secretions (hypodermins) stored in their midgut (Nelson and Weintraub, 1972) and undertake a long migration through the deep connective tissue of their host, where they are continuously exposed to immune effector mechanisms. Infestation of cattle with Hypoderma sp. has been shown to induce an immunological response characterized by an increase in serum immunoglobulin G levels with a peak when the first warbles become detectable on the back and drop once L3 have emerged (Sinclair and Wassall, 1983; Boulard, 1985; Pruett and Barrett, 1985; Panadero et al., 1997, 2002). However, there is not apparent correlation between antibody titres and resistance to Hypoderma infestation in cattle (Pruett et al., 1989; Panadero, 1996). Although detailed analyses have been performed on humoral responses involving total IgG responses to Hypoderma there is no information available with regard to the production of different immunoglobulin isotypes and subclasses. Cattle develop acquired resistance after repeated exposures to larval antigens (Gingrich, 1982). This resistance is recognized as an important factor in controlling Hypoderma populations and depends upon the number of previous exposures and the number of larvae infesting the host (Baron and Weintraub, 1987). Numerous studies have implicated the isotype of antibodies in the effective protection against diseases (Brown et al., 1999; Avramidis et al., 2002; Hagiwara et al., 2005; Almería et al., 2009; Piper et al., 2009). IgG subclasses in both the murine and human systems have been reported to be differentially regulated by cytokines, which act as switch and growth factors for B cells at different stages of maturation (Stavnezer, 1996). Cytokine control of antibody production in cattle may differ from the regulatory pathways, which have been described in the mouse and human (Estes, 1996). It is clear from studies on the T lymphocyte response that the Th1/Th2 paradigm, postulated by Mosmann et al. (1986) in a murine model, is less well defined in ruminants (Estes and Brown, 2002). It is also well established that Th1 and Th2 cytokines have an influence on the antibody production and isotype expression; for instance, recombinant bovine IL-4 up regulates the expression of IgM, IgG1 and IgE in vitro (Estes, 1996). Cytokine responses and lymphocyte subsets during larval penetration in cattle experimentally infested by H. lineatum have been determined in recent studies (Dacal et al., 2009, 2011). At this early phase of the infestation there is an increase of both Th1 (IFN-␥) and Th2-type cytokines (IL-4 and IL-10) suggesting a Th0 response. However, there is no information describing the subsequent phases of larval migration, especially during their residence in the oesophageal submucosa and after their arrival at the subcutaneous tissue of the back.
231
The aim of this study was to study the dynamics of antigen-specific antibodies (IgM, IgG, IgG1 and IgG2), T lymphocyte subsets (CD2, CD4 and CD8) and cytokine profiles (IFN-␥ and IL-4), and their possible relationships during the course of natural infestations by Hypoderma in cattle. 2. Materials and methods 2.1. Animals and blood samplings The trial was carried out on a beef farm with a history of hypodermosis. Identification of third instars (L3) obtained in previous infestations on the farm, showed the ˜ presence of H. lineatum. On this farm, located in A Coruna (Northwestern Spain), cows are raised in a semiextensive grazing system, allowing natural infestations by Hypoderma sp., whereas calves are maintained indoors until weaning (8–11 months of age) them they are destined for the slaughterhouse. They fed milk from their mothers and concentrates are gradually introduced in their diet. The herd received benzimidazole treatments twice a year, but drugs for lice or Hypoderma control were not used routinely and no fly control product was used during the summer months. Routine faecal examinations, using flotation and sedimentation techniques were negative to parasitic forms. Seven 1-year-old Rubia Gallega heifers were selected for this study on the basis of the presence of both anti-Hypoderma antibodies and the parasite antigen hypodermin C (HyC), as determined by an indirect (Panadero et al., 1997) and sandwich ELISA (Panadero et al., 2002), respectively. Those animals were in their 1st grazing year, thus it was considered that they were undergoing their first infestation by Hypoderma. All the animals were bled monthly (May 2007–May 2008) by caudal venipuncture, covering the entire endogenous cycle of H. lineatum in our region (Panadero, 1996; Panadero et al., 2007). Warbles were counted by manual palpation at monthly intervals. Results are expressed as the mean number of warbles per infested animal. Infested animals were treated subcutaneously with ivermectin (Ivertin® injectable, Laboratorios Calier, Spain) at the recommended dose (0.2 mg/kg p.v.) 2 months before the end of the study (March 2008). An uninfested control group, comprised by four Rubia Gallega calves, was also bled at monthly intervals. Control calves were born at the farm and were maintained indoors, avoiding their contact with the parasite. They were 3month-old at the beginning of the study and they remained at the farm until weaning (11 months of age). In consequence, control calves were only available for the first 8 months of the trial and their results are expressed as mean (SD) of the individual values obtained from May to December 2007. 2.2. ELISA protocols 2.2.1. Antibody detection Serum samples were processed by an indirect ELISA test described by Panadero et al. (1997) for IgG detection against the antigenic fraction hipodermin C (HyC).
L. Vázquez et al. / Veterinary Parasitology 184 (2012) 230–237
Abs (490 nm)
a
1,4
14
1,2
12
1,0
10
0,8
8
0,6
6
0,4
4
0,2
2
0,0
Warbles/infested animal
232
0 My
Jn
Jl
Au
St
Oc
Nv
Dc
Ja
Fb
Mr
Ap
My
Month
Abs (490 nm)
b
Control
1,4
14
1,2
12
1,0
10
0,8
8
0,6
6
0,4
4
0,2
2
0,0
Warbles/infested animal
IgG
0 My
Jn
Jl
Au
St
Oc
Nv
Dc
Ja
Fb
Mr
Ap
My
Month IgM
Control
Fig. 1. Kinetic of the antigen-specific IgG (a) and IgM (b) responses in cattle during natural infestations by Hypoderma sp. Bars represent the intensity of infestation. Vertical lines indicate standard deviation and horizontal line represent mean value of controls.
Horseradish peroxidase-conjugated sheep anti-bovineIgG1 (ShAB IgG1-HRP, 1/5000), ShAB IgG2 (1/500) and ShAB IgM (1/20000) were purchased from Serotec (Oxford, UK). Positive and negative control sera were introduced in every plate to normalize absorbance values. 2.2.2. Cytokine detection IL-4 and IFN-␥ serum levels were determined by sandwich ELISAs that specifically detect soluble cytokine proteins according to the protocols described in Dacal et al. (2009). Serum concentrations (pg/ml) were calculated according quadratic curves using recombinant bovine IL-4 and IFN-␥ (Serotec, Oxford, UK). 2.3. Flow cytometric analysis of lymphocyte subsets Lymphocytes were analyzed for expression of cellsurface differentiation antigens by direct immunofluorescence labelling. Briefly, fresh blood collected into sterile acid citric dextrose (ACD) tubes was incubated with fluorescein (FITC)-conjugated monoclonal antibodies against bovine CD2, CD4 and CD8 (Serotec, Oxford, UK) or an isotype control (Mouse IgG1 and IgG2a: FITC) at room temperature for 30 min. Then 2 ml of PBS/BSA 1% was added to each tube and were centrifuged at 500 × g for 10 min and the supernatant was discarded. Red cells were lysed
with 2 ml of lysis buffer BD FACSTM Lysing Solution (Beckton Dickinson). The mix was incubated at 25 ◦ C in agitation for 10 min and then centrifuged at 500 × g for 5 min and the supernatant discarded. Finally, the cells were resuspended with 400 l de PBS/BSA 1%. Labelled cells were detected in an Epics XL (Coulter Corporation, USA) cytometer. Data from 50,000 cells per sample were acquired using a 15 mW air-cooled argon ion laser with excitation wavelength of 488 nm. The results were analyzed with the System II program (Coulter Corporation, USA). Data are expressed as a percentage of positive cells. 2.4. Statistics Data were analyzed using the Statistical Package for Social Sciences (SPSS), Version 18.0.1 (SPSS Inc., Chicago, IL). Differences in the different parameters over time were studied by repeated measures ANOVA using the repeated contrast as a within-subject contrast to compare adjacent measures (P < 0.05). One sample t-test was used to determine whether the average values of the different parameters from control calves differed significantly from monthly values of primoinfested cows. Correlations between specific antibodies, lymphocyte subsets and cytokine concentrations were assessed using the Spearman rank correlation test.
Abs (490 nm)
a
233
1,4
14
1,2
12
1,0
10
0,8
8
0,6
6
0,4
4
0,2
2
Warbles/infested animal
L. Vázquez et al. / Veterinary Parasitology 184 (2012) 230–237
0
0,0
My
Jn
Jl
Au
St
Oc
Nv
Dc
Ja
Fb
Mr
Ap
My
Month
Abs (490 nm)
b
Control
1,4
14
1,2
12
1,0
10
0,8
8
0,6
6
0,4
4
0,2
2
0,0
Warbles/infested animal
IgG1
0 My
Jn
Jl
Au
St
Oc
Nv
Dc
Ja
Fb
Mr
Ap
My
Month IgG2
Control
Fig. 2. Kinetics of the subclasses IgG1 (a) and IgG2 (b) in cattle during natural infestations by Hypoderma sp. Bars represent the intensity of infestation. Vertical lines indicate standard deviation and horizontal line represent mean value of controls.
3. Results 3.1. Warble counts Warble appearance and the intensity of infestation are represented in Figs. 1–4. Four out of seven heifers presented cattle warbles with a mean of 11.2 (1.33 SD) warbles per infested animal (range 1–30 warbles). Warbles appeared in February, reaching a peak in this month. One month later the number of warbles had considerably decreased (1.3; 0.57 SD) and no nodules were observed in April, after the administration of a treatment. 3.2. Antigen-specific antibody responses IgG levels at the early stages of the infestation were relatively elevated (Fig. 1a), but they increased considerably once L1 arrived to the back, with a significant increment in March and a subsequent descent in May. Mean OD value for control calves was 0.31 (0.149 SD). One sample t-test showed significant differences between fixed mean IgG values from control group and primoinfested cattle from June to December 2007 and from February to April 2008. IgM maintained constant levels throughout the study (Fig. 1b). Mean OD value for control calves (0.11; 0.046 SD)
was significantly lower to that of infested cattle along the study. The evolution of the subclasses IgG1 and IgG2 are represented in Fig. 2. IgG1 profiles at the beginning of the infestation remained relatively constant, and they increased significantly in November, December and January. Once warbles appeared on the back, IgG1 levels started to decline. Infested animals showed higher OD values (P < 0.05) than the mean of controls (0.18; 0.162 SD) in July, August and from December to April. IgG2 profiles increased significantly in June, July and October, and decreased in November. Mean OD in controls for IgG2 (0.06; 0.025 SD) was significantly higher than in those infested in May, June, September, December and April. 3.3. Changes in circulating lymphocyte subsets Fig. 3 shows the evolution of circulating lymphocyte subsets. The percentage of CD2+ cells was relatively elevated at the beginning of the study (36.7%), and showed a significant increment in January, with a peak in February, coinciding with the apparition of warbles on the back. Control animals showed a mean percentage of CD2+ cells of 21.9% (5.17 SD); this value was significantly lower than those of the infested animals in May and October 2007.
234
L. Vázquez et al. / Veterinary Parasitology 184 (2012) 230–237 14
100
12
80
10 60
8 6
40
4 20
Warbles/infested animal
% Lymphocytes
a
2
0
0 My
Jn
Jl
Au
St
Oc
Nv
Dc
Ja
Fb
Mr
Ap
My
Month CD2
100
14 12
80
10 60
8 6
40
4 20
Warbles/infested animal
% Lymphocytes
b
Control
2
0
0 My
Jn
Jl
Au
St
Oc
Nv
Dc
Ja
Fb
Mr
Ap
My
Month CD4
100
14 12
80
10 60
8 6
40
4 20
Warbles/infested animal
% Lymphocytes
c
Control
2
0
0 My
Jn
Jl
Au
St
Oc
Nv
Dc
Ja
Fb
Mr
Ap
My
Month CD8
Control
Fig. 3. Systemic lymphocyte subpopulations, CD2 (a), CD4 (b), CD8 (c) in blood of cattle naturally infested by Hypoderma determined by flow cytometry. Bars represent the intensity of infestation. Vertical lines indicate standard deviation and horizontal line represents mean value of controls.
CD4 and CD8 percentages did not varied significantly along the study. Mean percentage for control calves was 10.9 (2.01 SD) and 7.3 (1.91 SD) for CD4 and CD8, respectively. Mean CD4 values did not differ from those of the infested animals along the trial, and for CD8 differences were only significant in May 2007. CD4/CD8 ratios were always higher than 1 throughout the infestation, with the highest ratio in October (1.8).
3.4. Evolution of circulating cytokines The evolution of serum concentrations of the cytokines IFN-␥ and IL-4 are represented in Fig. 4. IFN-␥ showed two significant drops in October and January. The concentration of this cytokine in control animals was 11.3 pg/ml (8.54 SD), and this value differed significantly from the concentration found in the infested group in January and May 2008.
L. Vázquez et al. / Veterinary Parasitology 184 (2012) 230–237
100
14 12
80
10 60
8 6
40
4 20
2
0
Warbles/infested animal
Concentration (pg/ml)
a
235
0 My
Jn
Jl
Au
St
Oc
Nv
Dc
Ja
Fb
Mr
Ap
My
Month IFN-γ
100
14 12
80
10 60
8 6
40
4 20
2
0
Warbles/infested animal
Concentration (pg/ml)
b
Control
0 My
Jn
Jl
Au
St
Oc
Nv
Dc
Ja
Fb
Mr
Ap
My
Month IL-4
Control
Fig. 4. Serum concentrations of IFN-␥ (a) and IL-4 (b) in pg/ml in cattle naturally infested by Hypoderma. Bars represent the intensity of infestation. Vertical lines indicate standard deviation and horizontal line represents mean value of controls.
Circulating IL-4 levels showed a significant decrease in January followed by an increase in February and May. Mean concentration for control animals was higher than that of the infested group in January and lower in May 2008. 3.5. Correlations between specific antibodies, lymphocyte subsets and cytokine concentrations IgM antibodies showed a strong negative correlation with CD2 ( = −0.358; P = 0.009) and with CD8 counts ( = −0.340; P = 0.014). IFN-␥ concentrations were negatively correlated with the IgG isotype ( = −0.218; P = 0.038) and with the IgG1 subclass ( = −0.219; P = 0.037), whereas IL-4 values were positively correlated with IgG2 subclass ( = −0.217; P = 0.038). 4. Discussion This study investigates the isotype pattern, lymphocyte subsets and cytokine profile during the course of natural infestations by Hypoderma as a contribution to increase the understanding of the pathways of stimulation of the immune system in this myiasis. Traditionally, antibody titres are not considered a reliable guide to the level of resistance against Hypo-
derma (Pruett et al., 1989; Panadero, 1996); however, their involvement in protection mechanisms has not been excluded and some vaccine studies aiming at inducing high levels of antibodies have been very promising (Pruett et al., 1987; Colwell, 2011). In our work, specific IgG antibody levels were similar to those reported in several other studies (Sinclair et al., 1984; Colwell and Baron, 1990; Panadero et al., 1997), with maximum levels coinciding with the presence of warbles on the back. The IgG1 subtype predominated over IgG2 during the entire life-cycle of the parasite, except in October (IgG1/IgG2 ratio 0.648). The maximum IgG1/IgG2 ratio was detected in January (3.8). IgG2 subclass showed an early elevation, which would coincide with the arrival of H. lineatum L1 to the oesophagus. Both subclasses decreased significantly when L3 began to leave the host. Our results suggest that Hypoderma infestation provokes an intense humoral response, characterized by an initial elevation of IgG2, which would be followed by a phase with a clear predominance of specific IgG1. In contrast, specific IgM remained constant throughout the study, although with higher levels than controls. According to Gold and Defranco (1994), this type of response would indicate a T-dependent antibody response, characterized by an extensive switching to immunoglobulin isotypes other than IgM; whereas an antibody response to T-independent antigens would be dominated by IgM. Despite the relatively
236
L. Vázquez et al. / Veterinary Parasitology 184 (2012) 230–237
weak IgM response, there was a negative correlation with CD2 and CD8 subsets. Nevertheless, the mechanisms of this relation remain to be elucidated. In our study CD2+ cells remained at low levels in the early stages of the infestation and showed a significant increase in the later phases of the endogenous cycle of the parasite. Similarly, López et al. (2005) observed a low lymphocytic perivascular infiltration in primary infested animals in the early stages of the infestation. In a previous study, Dacal et al. (2009) reported a local predominance of helper over cytotoxic T cells during the phase of larval penetration. In this study, we have confirmed that this predominance persists along the entire life-cycle of the parasite into the host. According to several authors (Broadmeadow, 1984; Bowles et al., 1992, 1994; Colditz et al., 1996), T CD4+ cells are also implicated in the immune response against Lucilia cuprina in sheep, whereas T CD8 cells are scarce in primary infestations. In murine systems, IL-4 and IFN-␥ reciprocally regulate isotype production directly via their actions on B cells and indirectly through T helper cell differentiation pathways leading to Th1 or Th2 responses (see review by Estes, 1996). Regarding the kinetics of IFN-␥, serum concentrations remained relatively constant during larval penetration and migration towards the oesophagus and they began to decline in October; this decrease continued until the end of the endogenous parasite life-cycle. Similarly, Dacal et al. (2009) did not observe significant variations of this cytokine during larval penetration in cattle experimentally infested by H. lineatum. On the contrary, Bowles et al. (1994) reported increasing levels of IFN-␥ after the infestation by L. cuprina. Serum concentrations of IL-4, a key regulator of humoral and acquired immune responses, did not show significant variations until the last phases of larval development. Thus, there was a significant but transient decline just before the arrival of L1 to subcutaneous tissues and an increment once warbles appeared on the back. Our results revealed a negative correlation between IFN-␥ serum concentrations and IgG and IgG1 antibodies, but surprisingly we did not find any relationship with IgG2, which has been reported to be up regulated by this cytokine (Snapper and Paul, 1987). Therefore, the negative correlation between IgG and IFN-␥ is mainly due to IgG1. In contrast with Estes (1996), who demonstrate that recombinant bovine IL-4 up regulated the expression of IgG1 in vitro, we found a positive correlation between the IL-4 and the IgG2 subclass. Once analyzed the evolution of the different parameters together it can be concluded that, as stated by Estes and Brown (2002), the polarisation of the immune response is a dynamic process with variations throughout the different phases of development of the parasite into the host. In our study we have observed that, in the early stages of the infestation (larval penetration and arrival to the oesophagus), there is a slight predominance of a Th1 response characterized by relatively elevated IgG2 and IFN-␥ levels. The dominance of this type of response would result in a stimulation of cellular responses against the parasite which may be effective in killing some of the entering larvae, resulting in a degree of resistance. In our study, only four out of seven
infested animals were palpation positive, although the intensity of infestation was elevated in comparison with that of reinfested cows (data not shown). Panadero et al. (2009) found that larval antigens are capable to combine the activation of cellular responses with an immunosuppressive effect, especially in naïve cattle. However, further studies are necessary to study the mechanisms implicated in the development of acquired resistance after successive reinfestations. Later, during the mobilisation of L1 from the oesophagus and their arrival to the back, the immune reaction is more intense with a clear predominance of a Th2 response, characterized by high levels of IgG1 and IL-4. According to Gingrich (1980), most larvae that die in resistant hosts may not be able to reach the oesophagus, i.e. they would die shortly after entering their host. Thus, the primary effectors of acquired resistance are functioning in the skin and/or the connective tissues along the migration route and non-existent or non-functional in the oesophagus and subsequent portions of the migration route that lead to larval encapsulation in the back. Acknowledgements We are very grateful to the Rubia Gallega breeder “Pepe ˜ for his colde Miralles” (Sobrado dos Monxes, A Coruna) laboration in the realization of this study. This research was supported by the Research projects AGL-2004-01827 and AGL-2009-08939. Authors also thank the Spanish Ministry of Education and Science for L. Vázquez and V. Dacal pre-doctoral FPU grants. References Almería, S., Nogareda, C., Santolaria, P., Garcia-Ispierto, I., Yániz, J.L., López-Gatius, F., 2009. Specific anti-Neospora caninum IgG1 and IgG2 antibody responses during gestation in naturally infected cattle and their relationship with gamma interferon production. Vet. Immunol. Immunopathol. 130, 35–42. Avramidis, N., Victoratos, P., Yiangou, M., Hadjipetrou-Kourounakis, L., 2002. Adjuvant regulation of cytokine profile and antibody isotype of immune responses to Mycoplasma agalactiae. Vet. Microbiol. 88, 325–338. Baron, R.W., Weintraub, J., 1987. Lymphocyte responsiveness in cattle previously infested and uninfested with Hypoderma lineatum (De Vill.) and Hypoderma bovis (L.) (Diptera: Oestridae). Vet. Parasitol. 21, 43–50. Boulard, C., 1985. Avantages de l’immunodiagnostic de l’hypodermose bovine établi par hemaglutination passive et par ELISA à partir du sérum et du lactosérum sur la numeration des varrons. Ann. Rech. Vet. 16, 335–343. Bowles, V.M., Grey, S.T., Brandon, M.R., 1992. Cellular immune responses in the skin of sheep infected with larvae of Lucilia cuprina, the sheep blowfly. Vet. Parasitol. 44, 151–162. Bowles, V.M., Meeusen, E.N., Chandler, K., Verhagen, A., Nash, A.D., Brandon, M.R., 1994. The immune response of sheep infected with larvae of the sheep blowfly Lucilia cuprina monitored via efferent lymph. Vet. Immunol. Immunopathol. 40, 341–352. Broadmeadow, M., 1984. Pathogenesis of blowfly strike in sheep. Wool Technol. Sheep Breed. 32, 28–32. Brown, W.C., McElwain, T.F., Palmer, G.H., Chantler, S.E., Estes, D.M., 1999. Bovine CD4+ T-lymphocyte clones specific for rhoptry-associated protein 1 of Babesia bigemina stimulate enhanced immunoglobulin G1 (IgG1) and IgG2 synthesis. Infect. Immun., 155–164. Colditz, I.G., Eisemann, C.H., Tellam, R.L., Mcclure, S.J., Mortimer, S.I., Husband, A.J., 1996. Growth of Lucilia cuprina larvae following treatment of sheep divergently selected for fleece rot and fly strike with monoclonal antibodies to T lymphocyte subsets and interferon gamma. Int. J. Parasitol. 26, 775–782.
L. Vázquez et al. / Veterinary Parasitology 184 (2012) 230–237 Colwell, D.D., Baron, R.W., 1990. Early detection of cattle grub infestation (Hypoderma lineatum DeVill. and H. bovis L.) (Diptera: Oestridae) using ELISA. Med. Vet. Entomol. 4, 35–42. Colwell, D.D., 2011. Hidden antigens from third instar Hypoderma lineatum: impact of immunization on larval survival in artificial infestations. Vet. Parasitol. 175, 313–319. Dacal, V., Colwell, D.D., López, C., Pérez, V., Vázquez, L., Cienfuegos, S., Díaz, ˜ P., Morrondo, P., Díez-Banos, P., Panadero, R., 2009. Local and systemic cytokine responses during larval penetration in cattle experimentally infested with Hypoderma lineatum (Diptera: Oestridae). Vet. Immunol. Immunopathol. 131, 59–64. Dacal, V., López, C., Colwell, D.D., Vázquez, L., Díaz, P., Morrondo, P., Díez, P., Panadero, R., 2011. Immunohistochemical characterization of inflammatory cells in the skin of cattle undergoing repeated infestations with Hypoderma lineatum (Diptera: Oestridae) larvae. J. Comp. Pathol. 145, 282–288. Drummond, R.O., 1987. Economic aspects of ectoparasites of cattle in North America. In: Leanning, H.D., Guerrero, J. (Eds.), The Economic Impacts of Parasitism in Cattle. Proceedings of the MSD AGVET Symposium, 23rd World Veterinary Congress. Montreal, pp. 9–24. Estes, D.M., 1996. Differentiation of B cells in the bovine. Role of cytokines in immunoglobulin isotype expression. Vet. Immunol. Immunopathol. 54, 61–67. Estes, D.M., Brown, W.C., 2002. Type 1 and type 2 responses in regulation of Ig isotype expression in cattle. Vet. Immunol. Immunopathol. 90, 1–10. Gingrich, R.E., 1980. Differentiation of resistance in cattle to larval Hypoderma lineatum. Vet. Parasitol. 7, 243–254. Gingrich, R.E., 1982. Acquired resistance to Hypoderma lineatum: comparative immune response of resistant and susceptible cattle. Vet. Parasitol. 9, 233–242. Gold, M.R., Defranco, A.L., 1994. Biochemistry of B lymphocyte activation. Adv. Immunol. 55, 221–295. Hagiwara, K., Tokuda, M., Baba, T., Yamanaka, H., Kirisawa, R., Tsuji, M., Ishihara, C., Iwai, H., 2005. The role of IFN-␥ in cattle infected with Theileria sergenti. Vet. Parasitol. 127, 105–110. López, C., Colwell, D.D., Panadero, R., Paz, A., Pérez, J., Morrondo, P., Díez, P., Cascallana, J.L., Santamaría, V., Bravo, A., 2005. Skin immune responses in cattle after primary and secondary infections with Hypoderma lineatum (Diptera: Oestridae) larvae. Vet. Immunol. Immunopathol. 108, 285–294. Mosmann, T.R., Cherwinski, H., Bond, M.W., Giedlin, M.A., Coffman, R.L., 1986. Two types of murine helper T cell clone. I. Definition according to profiles of lymphokine activities and secreted proteins. J. Immunol. 136, 2348–2357. Nelson, W.A., Weintraub, J., 1972. Hypoderma lineatum (De Vill.) (Diptera: Oestridae): invasion of the bovine skin by newly hatched larvae. J. Parasitol. 58 (3), 614–624. Panadero, R., 1996. Estudio epidemiológico y de la respuesta inmune rrente a antígeno de Hypoderma lineatum como aportación a la lucha
237
contra la hipodermosis bovina en Galicia. Tesis Doctoral, Facultad de Veterinaria, Universidad de Santiago de Compostela, 255 pp. ˜ P., 1997. Effect Panadero, R., López, C., Mezo, M., Morrondo, P., Díez-Banos, of early treatment with ivermectin and doramectin on the dynamics of antibody response in cattle naturally infested by Hypoderma lineatum and H. bovis. Vet. Parasitol. 73, 325–334. Panadero, R., López, C., Paz, A., Díez, P., Morrondo, P., 1998. Estudio sobre el desarrollo de las larvas esofágicas de Hypoderma lineatum y su relación con el tratamiento precoz de la hipodermosis bovina en el Norte de ˜ Vet. México 29, 155–160. Espana. ˜ P., Colwell, Panadero, R., López, C., Parra, F., Morrondo, P., Díez-Banos, D.D., 2002. Detection of circulating hypodermin C: an antigen capture ELISA for diagnosis of cattle grub (Diptera: Oestridae) infestations. Vet. Parasitol. 108, 85–94. Panadero, R., Fernández, M., Vázquez, L., López, C., Dacal, V., Cienfuegos, ˜ P., 2007. Occurrence and larval S., Diaz, P., Morrondo, P., Díez-Banos, growth of Hypoderma lineatum in the oesophagi in cattle from Northwest Spain: influence of geographical and climatic conditions. Med. Vet. Entomol. 21, 225–230. Panadero, R., Dacal, V., López, C., Vázquez, L., Cienfuegos, S., Díaz, P., ˜ P., 2009. Immunomodulatory effect of Morrondo, P., Díez-Banos, Hypoderma lineatum antigens: in vitro effect on bovine lymphocyte proliferation and cytokine production. Parasite Immunol. 31, 72–77. Piper, E.K., Jonsson, N.N., Gondro, C., Lew-Tabor, A.E., Moolhuijzen, P., Vance, M.E., Jackson, L.A., 2009. Immunological profiles of Bos taurus and Bos indicus cattle infested with the Cattle Tick, Rhipicephalus (Boophilus) microplus. Clin. Vac. Immunol, 1074–1086. Pruett, J.H., Barrett, C.C., 1985. Kinetic development of humoral antiHypoderma lineatum antibody activity in the serum of vaccinated and infested cattle. Southwest. Entomol. 10, 39–48. Pruett, J.H., Barrett, C.C., Fisher, W.F., 1987. Kinetics development of serum antibody to purified Hypoderma lineatum proteins in vaccinated and non-vaccinated cattle. Southwest. Entomol. 12, 79–88. Pruett, J.H., Fisher, W.f., Temeyer, K.B., 1989. Evaluation of purified proteins of Hypoderma lineatum as candidate immunogens for a vaccine against bovine hypodermiasis. Southwest. Entomol. 14, 363–374. Reina, D., Martínez-Moreno, F., Hernández, S., Navarrete, I., 1998. Chronobiology of bovine hypodermosis in Europe. In: Boulard, C., Sol, J.; Pithan, K., O’Brien, D., Webster, K., Sampimon, C. (Ed.), Improvements in the Control Methods for Warble Fly in Livestock. Brussels, European Commission, p. 56. Sinclair, I.J., Wassall, D.A., 1983. Enzyme-linked immunosorbent assay for the detection of antibodies to Hypoderma bovis in cattle. Res. Vet. Sci. 34, 251–260. Sinclair, I.J., Tarry, D.W., Wassall, D.A., 1984. Persistence of antibody in calves after an infection with Hypoderma bovis. Res. Vet. Sci. 37, 383–384. Snapper, C.M., Paul, W.E., 1987. Interferon-gamma and B cell stimulatory factor-1 reciprocally regulate Ig isotype production. Science 236, 944–947. Stavnezer, J., 1996. Antibody class switching. Adv. Immunol. 61, 79–146.