STUDIES ON GASTROINTESTINAL NEMATODES INFECTION IN SHEEP WITH SPECIAL REFERENCE TO HAEMONCHUS CONTORTUS

         Received: 17/1/2012

         Accepted: 29/2/2012

This study was carried out to investigate clinical, epidemiological and histopathological findings associated with gastrointestinal nematodes infection in sheep with special reference to Haemonchus contortus infection during the period from January, 2008 to June, 2010 at Gharbia Governorate, Egypt. The prevalence of gastrointestinal nematode among examined sheep was 62.38%. Trichostrongylus spp. was the most detected nematode. The highest prevalence was reported in females, during spring and in the age group over 2 years. The clinical findings observed in infected sheep were emaciation and pale mucous membrane. Infected sheep showed anemia and decrease in serum total proteins, albumin and globulins levels.

Introduction

            Gastrointestinal nematode (GIN) parasitism is arguably the most serious constraint affecting sheep production worldwide. Economic losses are attributed

contortus and Bunostomum trigonocephalum are responsible for specific clinical symptoms and great economic losses to small ruminant industry. Although Haemonchus species infect all ruminants, the severity of Haemonchus contortus infection is more pronounced in sheep where it is linked to severe anemia, diarrhea, loss of body weight and death (Agarwal and Banerjee, 2007). The objective of this study is to investigate the clinical, epidemiological, and histopathological findings associated with gastrointestinal nematodes in sheep at Gharbia Governorates, Epypt.

MATERIALS and METHODS

1. Animals:-

Three hundred and nineteen sheep belonging to 8 flocks of different age and sex (Table 1) were used in this study during the period from January, 2008 to June, 2010. All flocks were depending on grazing.

2. Clinical examination

All sheep used in this study were subjected to clinical examination according to.Kelly (1984).

3. Samples:-

3.1. Fecal samples

Fecal samples were examined macroscopically and microscopically using concentration floatation technique and the positive samples were subjected to fecal egg count, fecal culture, and larval identification according toSoulsby (1982).

3.2. Blood samples

Two blood samples were collected from each infected animal; one with anti coagulant to be used for hematological examination and the other without to be used for serum separation. These samples were collected via jugular vein puncture. Erythrocytic cell counts (RBCs),hemoglobin concentration (Hb) and Packed Cell Volume (PCV) were estimated according to Coles (1989). Moreover, MCV, MCH  and  MCHC  also

RESULTS

Prevalence and percentage of different gastrointestinal nematodes

Out of 319 examined sheep, 199 were found to be infected with gastrointestinal nematodes via parasitological examination representing 62.38%. (Figure 1). The highest infection was recorded by Trichostrongylus species was 59.25%, (Haemonchms 30%), followed by Strongyloides (13.79%) then Trichuris (0.94%) and Nematodirus (0.63%). Concerning the seasonal distribution of parasitic infestation, higher prevalence was recorded in spring (71.59%) followed by autumn (70.31%) then winter (68.91%) and finally summer (43.01%).

The prevalence of the recovered 3^rd stage larvae from fecal culture revealed that the predominant species in winter were Haemonchus (30%) followed by Bunstomum (20%) and Trichostrongylus species (20%). While, in spring were Haemonchus (30%) and Trichostrongylus species (30%) and in summer Bunstomum was the most predominant species (30%) followed by Haemonchus (20%) and Strongyloides (20%) then in autumn Haemonchus was the most predominant species (30%) followed by Trichostrongylus axei (20%).

to decreased production, costs of prophylaxis and treatment, and deaths of the infected animals. (Miller and Horohov, 2006).

Nematode species especially those fed   on      blood      such     as     Haemonchus

were    calculated.   Blood    serum    total proteins, albumin and globulinswere determined using commercial kits according to Doumas (1971) and Henry (1974).

4. Epidemiological investigations

Some epidemiological parameters associated with gastrointestinal nematodes infection were estimated according to Martin et al. (1987).

5. Post mortem examination:-

Five Haemonchus contortus infected sheep were slaughtered and their abomasa were examined grossly and histopathologically. Adult worms were collected for identification. Suitable portion of the infected abomasum, showing gross lesions was collected and fixed in 10% neutral buffered formalin solution to be used for histopathological examination.

5.1. Identification of adult worms from infected abomasa:-

Adult worms were collected from infected abomasa, washed in physiological saline, fixed in glycerin-alcohol then embedded in Canada-blasm on glass slides and identified according to Soulsby (1982).

5.2. Histopathological examination:-

The fixed infected abomasa were embedded in paraffin wax. Five microns thick paraffin sections were prepared and stained with Haematoxylene and Eosin (H&E) and examined microscopically according to Drurag and Wallington (1980).

6. Statistical analysis:-

Statistical analysis was carried out by using statistical soft ware program (GMP for windows version 5.1, SAS Institute, Cary, NC, USA). Differences between means at P < 0.05 were considered significant.

The prevalence of gastrointestinal nematodes in relation to animal age and sex was illustrated in Table (2 & 3).

Clinical findings:-

            Clinical findings were illustrated in Table (4).

Hematological findings:-

The hematological and serum biochemical changes that associated with gastrointestinal nematodes infestation in the examined sheep were illustrated in Table (5 and 6).

Post mortem findings:-

Gross lesions

Thickening, hyperemia and small ulceration in addition to petechial hemorrhage at the site of worm attachment were observed in infested abomasa. Abomasal contents were dark in color and the adult worms were present grossly.

Identification of the recovered worms

The detected worms were identified as Haemonchus spp.

Histopathological findings

Histopathological changes in abomasa infected with Haemonchus, were illustrated in Figures (2 - 6)

Locality

No. of animals

Sex and age

Male

Female

Under 1 year

1 year up to 2 years

Over 2 years

Total

Under 1 year

1 year up to 2 years

Over 2 years

Total

Kohafa

54

4

8

3

15

8

15

16

39

Elmohami

43

6

6

2

14

4

16

9

29

Kafrelzait

20

3

2

0

5

5

5

5

15

Kotor

20

7

0

3

10

7

0

3

10

Nawag

12

2

1

0

3

1

4

4

9

Sprbay

91

13

8

3

24

27

29

11

67

Tanta

70

40

1

1

42

6

18

4

28

Zefta

9

2

1

0

3

0

5

1

6

Total

319

77

27

12

116

58

92

53

203

Degree of infestation

Fecal egg count

(EPG)

RBCs

(mil/cmm)

Hb

(gm/dl)

PCV

(%)

MCV

MCH

MCHC

Low

(less than 500 Epg)

166.66±33.33*

4.6±0.12*

9.66±0.08

31.53±0.63

68.7±3.1

21.04±0.71

30.66±0.34

Moderate

(500 and 1000 Epg)

700±57.73*

4.3±0.1*

8.36±0.7

26.8±1.7*

62.22±2.87

19.39±1.34

31.12±1.12

Severe

(Over 1000 Epg)

4133.33±2535.9*

4.1±0.1*

7.13±1.16

22.66±4.06*

54.85±8.79*

17.27±2.49*

31.69±0.7

Negative

-ve

5.2±0.16

10.46±0.52

33.16±0.83

63±1.04

19.85±0.64

31.51±0.8

Degree of infestation

Fecal egg count

(EPG)

Serum total protein

(g/100ml)

Serum albumin

(g/100ml)

Serum globulin

(g/100ml)

Albumin globulin ratio

(A/G ratio)

(%)

Low

(less than 500 Epg)

166.66±33.33*

9.4±0.05*

3.22±0.02*

6.17±0.07*

0.52±0.01

Moderate

(500 and 1000 Epg)

700±57.73*

8.8±0.17*

3.06±0.12*

5.73±0.23*

0.53±0.03

Severe

(Over 1000 Epg)

4133.33±2535.9*

8.3±0.32*

3±0.17*

5.3±0.15*

0.56±0.01

Negative

-ve

11.2±0.2

3.6±0.14

7.53±0.33

0.49±0.03

Fig.2: Mononuclear cell infiltration in between mucosal glands in Haemonchus Controtus infested abomasa. H.&E. X = 400.

    Fig.3: Mononuclear cell infiltration in between lamina propria in Haemonchus contortus infested abomasa H.& E. X = 100.

Fig. 4: Hemorrhage at abomasal wall in Haemonchus contortus infested abomasa. H.&E. X=400.

   Fig. 5: Cross section of adult Haemonchus worm within the abomasal wall.             X = 100.

Discussion

Sheep is considered as one of the important sectors in livestock production. Sheep may be affected with many diseases which decrease production. Parasitic gastroenteritis is one of them causing many economic losses (Miller and Horohov, 2006).

Concerning the prevalence of parasitic nematodes, out of 319 examined sheep, 199 (62.38%) were proved to be infested with parasitic gastrointestinal nematodes via flotation concentration technique. Nearly similar prevalence was reported by El-Fayoumi (1989),(65.83%). Higher prevalence was reported by Al-Gaabary et al. (2007),(71.69%). Lower prevalence (42.66%) was reported by Abdel-wahed and Salem (1999). The variations between the prevalence in different studies may be attributed to the type of rearing, hygiene and control measures where, sheep raised on high protein diet developed resistance to parasitic gastroenteritis (Knox and steel, 1999). In addition, the changes in climatic condition may affect the degree of infection where rain may lead to increase infection with parasitic gastroenteritis.

Highest infestation was recorded by Trichostrongylus species (59.25%) and the lowest one recorded by Nematodirus (0.63%). Similar results were recorded by Hashem and El-Sayed (1997) and Al-Gaabary et al. (2007) who found that Trichostrongylus was the predominant species in percent of 46.4% and 64.92% respectively.

Our results differed from that recorded by Gharib (1998) who found that Trichuris ovis was the most common species of gastrointestinal nematode.

Concerning the seasonal distribution of parasitic infestation,the highestprevalence was recorded in spring(71.59%)    and     the    lowest one was recorded in summer (43.01%). Similar result obtained by Altaif and Issa (1983);   results may be attributed to pregnancy and lactation that cause rising in fecal egg count (Vlassoff et al., 2001).The rise in fecal egg count in female with specific physiological status may be attributed to relaxation of immunity and resistance (Valderrábano   et al., 2006).

Some parasitologically positive animals appeared clinically normal. The clinical signs were severe emaciation, easily detached wool, pale mucous membrane and diarrhea in addition to bottle jaw in 2 cases. Our results were similar to those obtained previously by Yacob et al. (2009)who noticed that the severity of the clinical signs was related to the intensity of infection.

These clinical signs may be attributed to the decrease in the levels of serum total protein, albumin and globulin which lead to bottle jaw (Radostits et al., 2010). Beside that there was significant decrease in serum calcium (Hasan et al., 1986) and alkaline phosphatase. Moreover, there was significant reduction in bone mineral density (Thamsborg and Hauge, 2001). All these factors may lead to poor growth rate and emaciation. In addition, ulceration in abomasum and inflammation in intestinal wall interfere with digestion and absorption leading to diarrhea, emaciation and detached wool (Radostits et al., 2010). Severe anemia recorded byYacob et al. (2009) caused pale mucous membrane.

The hematological changes associated with parasitic gastrointestinal nematode in sheep showed negative correlation between the degree of infestation and the levels of RBCs, Hb and PCV. There were significant decrease in levels of RBCs and PCV and a significant decreases in serum total protein, albumin and globulin levels. In addition, there were in all infested animals. These results agree with the results obtaind by Radostits et al. (2010). In addition, there was a significant decrease in levels of MCV and MCH in severely infected animals only. Our   results   nearly   similar   to    results

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who recorded that the peaks of worm egg counts occurred in spring and in autumn.

Our results differed from the results of Aly et al. (1994) who found lowest infestation during autumn and spring and Khalafalla et al. (2011) who reported lowest infestation during spring.

The prevalence of the recovered 3^rd stage nematode larvae from fecal culture revealed that the predominant species in winter, spring and autumn were Haemonchus (30%) while in summer Bunstomum (30%). Nearly similar results were obtained by Reynecke et al. (2009)who recorded that Haemonchus species was the predominant during wet seasons from October to March. Our results differed from that obtained by Horak (2003) who recorded Haemonchus species as the predominant nematodes in summer season.

Concerning age predisposition, parasitic gastrointestionl infestation was significantly different (P<0.05) among age groups, where the prevalence was higher in the animals over 2 years (78.46%) followed by animals from 1 to 2 years (72.27%) and lastly in that below 1 year age (45.93%). Similar results were recorded by Ramadan et al. (1992)who recorded highest worm burden in adult and old sheep. On the other hand, our results were different from that obtained byKhan et al. (2010)who found high nematode infestation in young animals than adults.Vlassoff et al. (2001)attributed these results to development and increase in immunity during increase of age. On the other hand Bonfoh et al. (1995)observed no relation between animal age and prevalence of parasitic gastroenteritis.

Concerning sex predisposition, significant variation was recorded within different sex where the prevalence rate in female animals was 75.86% and in male animals was 38.79%. Similar results were obtained by Khan et al. (2010). These obtained by Yacob et al. (2009) who found that low levelofinfestation with Haemonchus contortus leading to normochromic normocytic anemia. These results may be attributed to the effect of the hemolytic factor that released from adult Haemonchus contortus on the surface of sheep RBCs lead to hemolysis(Fetterer and Rhoads, 1998).Moreoversignificant decrease in serum total protein, albumin and globulins levels were recorded. This may be attributed to the inflammatory enteropathy that occurred in the gastrointestinal tract and, in turn, the alteration of the intestinal micro-circulation, permeability and motility leading to albumin lossing enteropathy (Nesheim, 1993).Moreover, Rhodes et al. (1978)claimed that albumin might serve as a nutrient for the growing parasites and this might be responsible for the recorded hypoalbuminemia.

Post mortem examination of abomasum of sheep infected with Haemonchus contortus revealed thickening, hyperemia and small ulceration in addition to petechial hemorrhage at the site of worm attachment. The abomasal contents were dark in color and the adult worm was noticed grossly. Histopathologically,  there were mononuclear cell infiltration in between mucosal glands and lamina propria beside necrosis and sloughing of mucosal epithelium and presence of adult worm. Similar results were previously obtained byHertzberg et al. (2000).

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