Preliminary association of coat colour types and tolerance to Haemonchus contortus infection in West African Dwarf sheep

Abstract

Reduction in livestock productivity from internal parasites remains a major problem including the roundworm Haemonchus contortus. Treatment is rather expensive in most resource poor regions of the world, and the use of genetically resistant genotypes have been advocated. Our preliminary study investigated the association of coat colour types and tolerance to H. contortus infection in West African Dwarf (WAD) sheep. Twenty-four WAD sheep (12 males and 12 females) representing five coat colour types were orally dosed with 1000 H. contortus third-stage infective larvae, and data were collected over a 3-week period on initial and post-infection body weight (IBW and PIBW), packed cell volume (PCV), total serum proteins (TSP), albumin (ALB), globulin (GLB) and faecal egg counts (FEC). Coat colour was significantly associated with tolerance to H. contortus infection (p<0.05). Brown mouflon (A^mabbSs) sheep showed the highest mean PIBW, PCV and GLB (15.16±1.76 kg, 30.67±3.53% and 31.17±0.54 g/dl), respectively, while brown sheep with extensive white markings (aabbss) had the highest mean TSP and ALB value (73.40±2.02 g/dl and 44.47±0.49 g/dl), respectively. Badger face (A^bfaB-S-) sheep had the least mean values of PIBW, PCV, TSP, ALB, GLB and the highest FEC. There were no significant associations with sex and its interactions with coat colour on post-infection performances of WAD sheep in this study. Our results suggest that brown sheep can tolerate H. contortus infection better than other coat colour types, which may be due to the linkage between coat colour and resistance loci, linkage disequilibrium effects or resulting from pleiotropic effects deserving further study.

Keywords:

• coat colour gene
• tolerance
• Haemonchus contortus
• infection
• sheep

Introduction

Sheep production is considered to be an important economic activity in various regions of the world for meat, wool and hide (McManus et al. 2010). Endoparasitic infections represent major constraint in sheep production all over the world (Molento, 2009). Internal parasites reduce feed intake and nutrient absorption, consequently reducing growth rate by up to 30% or more (Perez et al. 2003). In addition, endoparasitism according to Ademola et al. (2004) may also result in reduced productivity, anoestrus, abortion, death of young animals and adults alike from infections of helminths and endoparasites such as roundworms, liver flukes, schistosomes and tapeworms. The roundworm Haemonchus contortus (with common names such as red stomach worm, wire worm or barber's pole worm) is one of the most serious infections in the tropics (Gatenby, 2002). H. contortus is prevalent in tropical and subtropical parts of the world and is also important in the temperate regions, especially during warm and wet conditions of spring and summer. H. contortus is a very prolific parasite which sucks blood leading to anaemic conditions and probable death (Vanimisetti et al. 2004). Pathogenic effects of the infection can range from depression in growth rate, diarrhoea, anaemia, to death depending on the severity. Mandonnet et al. (2005) reported the impact of helminth infestation including H. contortus on the poor productivity of Creole goats in the humid tropics.

Anti-helminthic resistance in several livestock species has emerged in many parts of the world (Coles et al. 2005; Waller et al. 2006) and the possible use of genetically resistant genotypes as an alternative and sustainable means of helminths control have been advocated (Vanimisetti et al. 2004). Gruner et al. (2003) observed that Barbados Black Belly sheep had higher resistance to H. contortus than the INRA401 breed. Amarante et al. (2006) reported that the relative resistance of Sanata sheep was superior to that of Suffolk and Ile de France sheep in Brazil. Chaudary et al. (2007) also reported that hairy (Jattal) goats and Salt-Range (Latti) sheep breeds in Pakistan exhibited significantly better resistance to H. contortus compared to other breeds from reduced log faecal egg count (FEC), higher packed cell volume (PCV) and haemoglobin levels. This project is a preliminary study of association of coat colour types and tolerance to H. contortus infection in West African Dwarf (WAD) sheep found in Nigeria.

Materials and methods

The protocol for the experiment was approved by the Institutional Animal Use and Care Committee of the University of Agriculture, Abeokuta (UNAAB), Nigeria. The experiment was carried out at the sheep unit of the Teaching and Research Farm of UNAAB (latitude 7° 10′ and longitude 3° 2′ E). The research location is situated in Odeda Local Government Area of Ogun State, in southwestern Nigeria, having ambient temperatures ranging from 28°C in December to 36°C in February with relative humidity of 82% and the average annual rainfall of 6.77 cm while the vegetation represents an inter-phase between the tropical rainforest and the derived savannah.

This experiment was conducted between July and October 2007, because of the availability of H. contortus worms during this period of the year. The average recorded rainfall for July, August, September and October is 12.19, 11.77, 11.81 and 9.22 cm, respectively. Twenty-four WAD sheep (12 males and 12 females) with different coat colour types under the same management system were used for the experiment. The coat colour types were solid black (aaB-S-), brown with extensive white markings (aabbss), brown mouflon (A^mabbss), badger face (A^bf-B-Ss or A^bf-bbSs) and black with extensive white markings (aaB-ss). They were separated into six animals (three female and three males) per group for badger face (BF) and black and four per group (two males and two females) for the remaining groups.

Coat colours were classified according to Adalsteinsson (1970, 1974) and Adalsteinsson et. al. (1994). The female sheep used were all primiparous and all animals were within the age range of 12–13 months. Preliminary analyses on worm infestation revealed that all of them had some level of Monieza spp., Fasciola spp., Fascioloides magna and H. contortus infection before the experiment started, because they were selected from groups of grazing animals. All the animals were dewormed at the start of the experiment using a broad spectrum antihelmintic (Albendazole®) at a dosage of 7.5 mg per kg of body weight according to manufacturer's protocol. This was followed by 3 weeks withdrawal period before the commencement of the experiment to prevent carryover effect. Animals were managed intensively with zero grazing involving provision of clean sun-dried grasses. Clean water was provided ad libitum. The grasses used for feeding were Panicum maximum and Pennisetum purpureum harvested from surrounding areas where livestock are not grazed. Supplemental concentrate consisted of maize (15%), wheat offal (45%), palm kernel cake (30%), oyster shell (3.5%), bone meal (5%), salt (1%) and multivitamin premix (0.5%). All sheep were challenged with equal dose of H. contortus larvae with approximately 1000 third stage larvae by drenching. This was repeated after 7 days followed by 3 weeks of data collection. Pre-infection body weight of each animal was taken. Faecal sample was collected directly from the rectum, and the FEC in egg per gram was determined for each animal using the McMaster counting chamber according to the method described by Hansen and Perry (1994).

About 4 – 5 ml of blood was collected through jugular venipuncture from each animal out of which 2 ml was dispensed into clean bottle containing Sodium EDTA. The rest were allowed to clot for sera collection. The uncoagulated blood was used to determine PCV and haematocrit count according to the methods described by Edington and Gillies (1981). Total serum proteins (TSP), albumin (ALB) and globulin (GLB) were determined from sera separated from the clotted blood according to methods described by Varley et al. (1980). Briefly, an aliquot of 0.1 ml was pipetted into a test tube and 2.9 ml water was added and a blank was set up with 3.0 ml distilled water. To each tube, 3.0 ml of working biuret reagent was added, and all the tubes were incubated in a 37°C water bath for 10 minutes. Readings were taken with a spectrophotometer at 540 nm. TSP of each sample was calculated from the formula:

Serum albumin was estimated using the bromocresol purple method of Varley et al. (1980). To 4.0 ml of bromocresol purple was 0.02 ml each of the test solution and the standard (ALB solution of known concentration) and 4.0 ml of bromocresol purple solution was used as blank. The content of each tube was mixed and left at room temperature for 10 min at pH 4.2±0.05. After 10 min, the test solution was read at a wavelength of 640 nm in a spectrophotometer set to zero with the blank solution.

Post-infection body weight, PCV, TSPs, ALB and GLB were taken weekly on each animal for a period of 3 weeks. Faecal samples were collected and FECs were determined twice in a week for a period of 3 weeks. The animals were later dewormed using a broad antihelmintic (Albendazole) at dosage of 7.5 mg/kg body weight. They were also treated with iron injection to recover from anaemia.

Statistical analysis

Faecal egg counts were not normally distributed and, therefore, a set of logarithmic transformation (log₁₀) was applied to the data and the resulting transformed variables were tested for normality with the univariate procedure of SAS (2010). These transformed data were used in all subsequent analysis. All data obtained were analysed using PROC MIXED of SAS (2010) with fixed effects of coat colour, sex and individual animals treated as random effects.

Results and discussion

Association of coat colour types and tolerance traits

The least squares mixed model analysis of variance for all the traits studied are presented in Tables 1–3. Generally, the difference among coat colour type was not significant in the first week and was only significant post-infection for FEC. However, in the second and third week post-infection, coat colour types became significant (p<0.01) resulting in reduction in the performance of animals with certain colour types. Coat colour was significantly (p<0.01) associated with body weights. Although brown mouflon (BM) sheep had the least variation in initial body weight, BF sheep had the highest variation in mean body weight throughout the 3-week experimental period. Table 4 shows the least square means of body weight according to coat colour types. The reduction in body weight of susceptible sheep was in agreement with the report of Vanimisetti et al. (2004).

Table 1. F values from mixed model analysis of variance of post-infection body weight changes and packed cell volume (PCV) in West African Dwarf sheep.

		Body weight changes (kg)				PCV (%)
Sources of variation	DF	Week 1	Week 2	Week 3	TPL	Week 1	Week 2	Week 3	TPL
Coat colour	4	89.83ns	113.65*	167.38*	206.41**	71.98ns	145.11*	167.07*	197.22*
Sex	1	82.37ns	63.33ns	54.38ns	81.91ns	44.27ns	59.08ns	47.12ns	58.11ns
Coat colour×sex	4	17.68ns	11.32ns	79.91ns	11.39ns	29.86ns	12.74ns	32.14ns	19.01ns
TPL, Total post-infection loss; ns, not significant.
*p<0.05.
**p<0.01.

Table 2. F values from mixed model analysis of variance of post-infection total serum protein (TSP) and serum albumin in West African Dwarf sheep.

		TSP (g/l)				Serum albumin (g/l)
Sources of variation	DF	Week 1	Week 2	Week 3	TPL	Week 1	Week 2	Week 3	TPL
Coat colour	4	99.87ns	122.61*	147.48*	186.41**	61.91ns	125.21*	144.07*	167.12*
Sex	1	68.32ns	67.31ns	54.31ns	71.92ns	42.27ns	79.08ns	57.11ns	48.21ns
Coat colour×sex	4	37.48ns	21.32ns	69.92ns	17.39ns	19.86ns	22.14ns	22.44ns	18.91ns
TPL, Total post-infection loss; ns, not significant.
*p<0.05.
**p<0.01.

Starting in the second week, post-infection PCV was significantly (p<0.01) higher when compared to the pre-infection data. The PCV data was highest in BM sheep throughout the experimental period, but the difference observed between BM and brown with extensive white markings (BRW) during the second and third week of infection was not significant as indicated in Table 5. Black sheep with extensive white markings (BLW) had a mean PCV value that is significantly lower than solid black coloured (SBC) sheep during the second and third week of infection. BF sheep had the least value during the experiment. Although there was a general decline in the PCV value of all the animals challenged with H. contortus from week 1 to week 3, the level of decline varied significantly (p<0.01) within the different colour groups. This was in agreement with Rajguru et al. (2002) who reported a significant decrease in the PCV in goats parasitised with H. contortus. Lower PCV values for animals suffering from haemonchosis have been reported (Ejlertsen et al. 2006; Fakae et al. 2004; Vanimisetti et al. 2004; Notter et al. 2003).

Table 3. F values from mixed model analysis of variance of post-infection serum globulin and faecal egg count (FEC) in West African Dwarf sheep.

		Serum globulin (g/l)				FEC
Sources of variation	DF	Week 1	Week 2	Week 3	TPL	Day 5	Day 7	Day 10	Day 14	Day 17	Day 21
Coat colour	4	69.77ns	132.11*	127.78*	186.41**	51.41ns	85.71*	104.07*	97.13*	99.74*	104.32*
Sex	1	58.12ns	76.37ns	64.21ns	57.91ns	35.21ns	49.18ns	37.11ns	38.11ns	21.09ns	36.11ns
Coat colour×sex	4	35.41ns	28.13ns	19.93ns	16.37ns	9.86ns	17.14ns	12.44ns	18.31ns	11.77ns	17.66ns
Notes: Means in the same column with the different superscripts are significantly different (p<0.05) for coat colour and sex.
TPL, Total post-infection loss; ns, not significant.
*p<0.05.
**p<0.01.

Table 4. Least square means of post-infection body weight (kg) as influenced by coat colour and sex.

	Initial	Week 1	Week 2	Week 3	Total post-infection loss
Coat colour
Badgerface (A^bfaB-S-)	15.20	12.58±0.19^a	11.17±0.11^b	9.75±0.18^b	5.42±0.15^b
Black with extensive white (aaB-ss)	15.30	13.34±0.13^a	12.17±0.08^b	11.08±0.17^ab	4.17±0.11^ab
Black (aaB-S-)	14.80	13.41±0.17^a	12.33±0.15^b	11.67±0.18^ab	3.17±0.14^ab
Brown mouflon (A^mabbS-)	15.70	14.43±0.18^a	14.07±0.01^a	13.66±0.16^a	2.05±0.21^a
Brown with extensive white (aabbss)	14.90	14.00±0.19^a	12.50±0.19^b	12.85±0.16^a	2.06±0.18^a
Sex
Male	16.90	16.17±0.20^a	14.83±0.14^a	13.79±0.14^a	4.08±0.19^a
Female	12.70	11.83±0.21^b	10.79±0.19^b	10.12±0.17^b	2.54±0.16^b

Table 6 shows the least square means for post-infection TSP by coat colour type. BRW sheep had the highest TSP in weeks 1 to 3 though not statistically different from BM sheep. SBC had a significantly higher mean value than BLW during the 3 weeks. Nevertheless, there was a general reduction in TSP but the extent of reduction was influenced by coat colour.

Table 5. Least square means for post-infection packed cell volume (%) as influenced by coat colour and sex.

	Initial	Week 1	Week 2	Week 3	Total post-infection loss
Coat colour
Badgerface (A^bfaB-S-)	34.16	29.33±0.24^a	24.67±0.12^c	20.50±0.28^c	13.67±0.16^c
Black with extensive white (aaB-ss)	33.83	30.50±0.23^a	25.83±0.25^c	24.17±0.40^c	9.67±0.22^b
Black (aaB-S-)	34.50	31.17±0.29^a	28.33±0.16^b	26.67±0.36^b	7.83±0.17^b
Brown mouflon (A^mabbS-)	35.00	33.33±0.15^a	32.00±0.16^a	30.67±0.33^a	4.33±0.17^a
Brown with extensive white (aabbss)	35.00	32.33±0.33^a	31.67±0.31^a	30.33±0.28^a	4.67±0.18^a
Sex
Male	33.50	30.00±0.36^a	26.58±0.33^a	24.50±0.39^a	9.00±0.16^a
Female	35.25	31.92±0.34^a	28.57±0.29^a	26.42±0.48^a	8.83±0.15^a
Note: Means in the same column with the different superscripts are significantly different (p<0.05) for coat colour and sex.

Coat colour type significantly differed for post-infection ALB levels (Table 7). BRW had the highest mean value in weeks 2 and 3, respectively. The difference in the mean values of BM and BRW sheep was significant over three-week period. The mean value for BLW sheep was significantly lower than SBC sheep over the entire experimental period. The difference in the mean values of BF and BLW was not significant in the weeks 1 and 2 but was significant in week 3. BF sheep had the least mean value in the three weeks of the research. BRW sheep had the highest value during the three weeks, while BF sheep had the least value (Table 7). BLW had a significantly lower GLB value than the SBC sheep in weeks 2 and 3 (Table 8).

Table 6. Least square means for post-infection total serum protein (g/l) as influenced by coat colour and sex.

	Initial	Week 1	Week 2	Week 3	Total post-infection loss
Coat colour
Badger face (A^bfaB-S-)	81.60	62.02±0.40^a	52.62±0.21^c	48.08±0.47^c	33.55±0.14^a
Black with extensive white (aaB-ss)	81.30	67.77±0.22^a	62.22±0.28^b	56.95±0.13^bc	24.33±0.11^b
Black (aaB-S-)	80.30	72.48±0.19^a	66.50±0.33^ab	63.45±0.27^b	16.85±0.15^bc
Brown mouflon (A^mabbS-)	81.80	76.77±0.24^a	73.20±0.23^a	70.13±0.21^a	11.70±0.28^c
Brown with extensive white (aabbss)	78.60	76.87±0.37^a	75.1±0.26^a	73.40±0.12^a	5.17±0.26^d
Sex
Male	81.50	68.32±0.13^a	61.25±0.36^a	57.68±0.41^a	23.82±0.14^a
Female	80.20	71.22±0.22^a	66.49±0.37^a	62.45±0.39^a	17.77±0.23^a
Note: Means in the same column with the different superscripts are significantly different (p<0.05) for coat colour and sex.

There was a progressive increase in the FEC from days 5 to 21 (Table 9), however this increase significantly (p<0.01) differed among coat colour types from day 7. BRW had the least mean value of FEC. The difference in the mean value for BM and BRW sheep was not significant throughout the experiment whereas BF sheep had the highest FEC. There was a weekly decline in TSP, ALB and GLB in all infected sheep, confirming the report of Rajguru et al. (2002) of significant decrease in the TSP of goats parasitised with H. contortus and the reduction in body weight of the susceptible sheep was in agreement with the report of Vanimisetti et al (2004).

Table 7. Least square means for post-infection serum albumin (g/l) as influenced by coat colour and sex.

	Initial	Week 1	Week 2	Week 3	Total post-infection loss
Coat colour
Badger face (A^bfaB-S-)	47.20	38.03±0.56^a	30.65±0.37^b	26.77±0.29^c	20.45±0.19^a
Black with extensive white (aaB-ss)	45.50	38.05±0.32^a	33.63±0.28^b	30.97±0.21^bc	14.55±0.42^ab
Black (aaB-S-)	46.40	42.05±0.36^a	38.78±0.22^ab	35.52±01.14^b	10.88±0.33^b
Brown mouflon (A^mabbS-)	45.00	43.27±0.28^a	40.70±0.10^a	38.97±0.08^a	6.07±0.28^bc
Brown with extensive white (aabbss)	47.50	46.57±0.35^a	44.97±0.26^a	44.47±0.19^a	3.07±0.22^c
Sex
Female	46.10	41.52±0.33^a	37.77±0.34^a	35.54±0.30^a	14.12±0.22^a
Male	46.60	40.00±0.16^a	35.17±0.28^a	31.94±0.22^a	11.10±0.24^a
Note: Means in the same column with the different superscripts are significantly different (P < 0.05) for coat colour and sex.

Sex differences

There was no significant sex differences in the traits studied, although males lost more weight than their female counterparts during the experiment. The results obtained for PCV, TSP, ALB, GLB and FEC followed a similar trend where neither of the sexes tolerates H. contortus better than the other. This observation is contrary to the report of Chauhan et al. (2003) in Barbari and Jamunapari goats, where they observed that Jamunapari males had higher FEC than the females while the PCV of male Barbari goats were significantly lower than that of their female counterparts when exposed to natural infection of H. contortus. Differences in these reports could however be attributed to the length of the study. The duration of the study reported by Chauhan was longer than this current study. Nakanishi et al. (1989) also demonstrated that testosterone reduced response of lymphocytes, macrophages and eosinophils to primary infection of C57BL/6 mice by Brugia pahangi consequently reducing resistance. Nelson et al. (2007) indicated that the sex of mice had no effect on Strongyloides venezuelensis in mice. The interaction between coat colour types and sex of the sheep did not significant in this study.

Table 8. Least square means for post-infection serum globulin (g/l) as influenced by coat colour and sex.

	Initial	Week 1	Week 2	Week 3	Total post-infection loss
Coat colour
Badgerface (A^bfaB-S-)	34.10	26.58±0.44^a	22.63±0.38^b	19.82±0.18^b	14.32±0.25^a
Black With extensive white (aaB-ss)	33.50	27.00±0.33^a	23.78±0.27^b	20.65±0.31^b	12.87±0.23^a
Black (aaB-S-)	34.00	30.32±0.21^a	27.70±0.34^ab	25.88±0.22^ab	8.10±0.17^b
Brown mouflon (A^mabbS-)	37.50	34.67±0.25^a	32.97±0.23^a	31.17±0.24^a	6.33±0.24^b
Brown with extensive white (aabbss)	31.00	29.67±0.28^a	29.17±0.17^a	28.70±0.15^a	2.30±0.40^c
Sex
Female	33.00	28.51±0.12^a	26.42±0.28^a	24.77±0.36^a	11.60±0.31^a
Male	35.00	29.52±0.23^a	25.99±0.22^a	23.37±0.19^a	8.19±0.18^a
Note: Means in the same column with the different superscripts are significantly different (p<0.05) for coat colour and sex.

Conclusions

This study demonstrated significant association of coat colour types and tolerance to H. contortus infection in WAD sheep. Badgerface coat colour type was very susceptible to parasite infestation. Badgerface sheep showed the least tolerance while BM sheep showed the highest tolerance to the parasite. This preliminary assessment will benefit from a larger study along with molecular analysis to better unravel the basis of these results. This will contribute to our understanding of the biology of tolerance, susceptibility and resistance as it relates to coat colour types in sheep.

Table 9. Least square means for post-infection log transformed faecal egg count as affected by coat colour and sex.

	Initial	Day 5	Day 7	Day 10	Day 14	Day 17	Day 21
Coat colour
Badgerface (A^bfaB-S-)	0	7.68±0.13^a	8.00±0.06^a	8.28±0.10^a	8.54±0.12^a	9.08±0.12^a	9.29±0.09^a
Black with extensive white (aaB-ss)	0	6.99±0.17^a	7.42±0.13^a	7.57±0.13^a	7.95±0.15^a	8.31±0.09^ab	8.69±0.10^ab
Black (aaB-S-)	0	6.34±0.18^a	6.91±0.11^a	7.34±0.13^ab	7.94±0.08^a	8.31±0.04^ab	8.58±0.12^ab
Brown mouflon (A^mabbS-)	0	5.29±0.01^a	5.20±0.32^b	6.56±0.20^b	7.41±0.09^b	7.58±0.15^b	7.81±0.12^b
Brown with extensive white (aabbss)	0	0^b	4.84±0.23^b	6.71±0.10^b	7.18±0.13^b	7.58±0.16^b	7.83±0.26^b
Sex
Male	0	7.08±0.26^a	6.99±0.34^a	7.57±0.20^a	8.09±0.15^a	8.38±0.19^a	8.74±0.18^a
Female	0	6.73±0.23^a	6.68±0.35^a	7.34±0.18^a	7.78±0.14^a	8.26±0.14^a	8.45±0.15^a
Note: Means in the same column with the different superscripts are significantly different (p<0.05) for coat colour and sex.