MATERIALS AND METHODS

Study area: The study was conducted from January to December 2017 in the Abattoir of Ngaoundere located within the following geographical limits latitude 7°-8° North and longitude 13°-14° East where approximately 55 cattle are slaughtered daily and most of them originate from the Vina division (with records on Trypanosoma spp., infection rates and its mechanical vectors) in the Adamawa Plateau and Mayo-Rey division (with records on mechanical vectors and Trypanosoma spp., occurrence) in the North region of Cameroon¹². The main breeds of cattle encountered in the abattoir include Red Fulani, Gudali×White Fulani, Charolais and Gudali¹².

Study population and sample size determination: The study population consisted of cattle slaughtered at the Ngaoundere Municipal Abattoir.

The sample size was determined using the following formula⁹:

Where:

An estimated prevalence of 50% was used with a precision level of 5% resulting in a minimum sample size of 384 cattle. The sample size was increased to 739 cattle far above the estimated value to avoid sampling errors.

Blood collection from cattle: The animals were randomly selected based on their availability and the consent of the owners. After restraining the animals, approximately 3 to 4 mL of blood was collected through the jugular vein into a tube containing Ethylene Diamine Tetra Acetic Acid (EDTA) anticoagulant. The tubes were labelled with the animal number, breed, sex, age, body condition score and origin.

Determination of breed, age and body condition score of animals: The cattle breed was determined using phenotypic characteristics. Age was determined by examining the dentition and horn rings. The animals were grouped into 3 age groups including young (<3 years), adults (3-8) and aged (>8 years). The BCS was established on a scale of 0-5, after examining the animal by palpation of the lumbar region as well as by assessing its general appearance following the method proposed by Mamoudou et al.¹³. The animals were divided into 3 groups according to their BCS: Bad (0-2), average (3) and good (4-5). The origin of the animals was the village or division where they were purchased.

Parasitological testing of cattle blood: The collected blood samples were transported to the laboratory in a cooler containing ice packs. Parasitological analysis was conducted within 5 hrs of collection using the Murray or Buffy Coat Method¹⁴. Briefly, the anticoagulant-containing capillary tubes (EDTA) were filled with 4/5 blood. The tubes were sealed with plasticine at one end and placed in the rotor of the microcentrifuge (Hettich HAEMATOKRIT). The blood was centrifuged at 12,000 rpm for 5 min.

Determination of the Packed Red Blood Cell Volume (PCV): The PCV was measured using the hematocrit reader (Hawksley Microhematocrit Reader^®). Animals with a PCV less than or equal to 24% were considered anaemic¹¹.

Microscopic identification of trypanosomes and determination of parasitaemia: The buffy coat was placed on a slide with a coverslip and examined microscopically (Micron optic^®) using the ×40 magnification. The determination of parasitaemia was conducted using the score of +1 to +6, corresponding to the number of trypanosomes per mL of blood.

Data analysis: The prevalence was obtained by using the following formula⁹:

Statistical analysis was conducted using the XLSTAT version 19.4 statistical software. The Chi-square test and the Kruskal-Wallis test were used to compare prevalence rates with associated risk factors. The degrees of significance of all statistical tests were kept at p<0.05.

RESULTS

Prevalence with the origin of slaughtered cattle in Ngaoundere Abattoir: The 739 cattle were sampled, including 412 in the dry season (January to March) and 327 in the rainy season (May to June). These animals originated from 14 cattle markets notably Dang, Dibi, Djalingo, Galdi, Likok, Mbe, Ngaoundere, Tchabal and Tello located in the Vina division and others from Gop Rey, Mbaka, Touboro, Vogzom and Yoko, found in the Mayo-Rey division. About 55% of these animals originated from the Mayo-Rey Division (n = 406) and 45% from the Vina division (n = 333). The prevalence concerning the divisions of origin indicated that higher cases were encountered in animals from Mayo-Rey than those from the Vina division.

Prevalence of bovine trypanosomiasis at the Ngaoundere Abattoir: Of the 739 cattle sampled, parasitological examination revealed 94 positive samples with parasitological prevalence rate of 12.72% (95% CI: [10.32-15.12%]).

Trypanosoma species microscopically typed: The trypanosome species encountered included Trypanosoma congolense, Trypanosoma brucei, Trypanosoma vivax and their mixed infections. A predominance of Trypanosoma congolense (43.62%:41/94) was observed, followed by Trypanosoma brucei (36.17%:34/94) and finally Trypanosoma vivax (12.76%:12/94). In addition to these monospecific infections, mixed infections were recorded, including Trypanosoma congolense +Trypanosoma brucei (5.32%:5/94), Trypanosoma congolense +Trypanosoma vivax (1.06%:1/94) and Trypanosoma congolense +Trypanosoma brucei +Trypanosoma vivax (1.06%:1/94). This difference between trypanosome species infection rates was significant (p<0.05).

Association of Trypanosoma spp. and parasitaemia on number of cases: The number of infected animals depended on the Trypanosoma infection states (monospecies and polyspecies) in Table 1. For the trypanosome species frequency, Trypanosoma brucei recorded high number of cases than other species with a non-statistically significant difference (p = 0.458).

The highest number of cases was recorded in animals with the lowest parasitaemia (Table 1). Concerning parasitaemia of animals, a low trypanosome load (10²-10⁴) was recorded for most of the animals (80 in total).

Prevalence concerning season: The parasitological prevalence of bovine trypanosomiasis with season showed that high cases were diagnosed in the rainy season (15.60%) than in the dry (10.44%) with a statistically significant difference (p<0.05) in Table 2.

Prevalence with breed, sex, age and season: In the dry season, it was noticed that the bokolo breed recorded the highest prevalence rate (33.33%) with a non-statistically significant difference (p = 0.487). In this same period, male (13.67%) cattle recorded a high prevalence rate with no statistically significant difference (p = 0.463). The age cohort with high prevalence was 3-8 years (11.11%) with no statistically significant difference (p = 0.701). For the rainy season, the Gudali cattle breed recorded a high prevalence rate (19.28%) with no statistically significant difference (p = 0.496). Male (21.43%) cattle during the rainy season had high infection rate than their female (15.05%) counterparts with no statistically significant difference (p = 0.374) and the age cohort with a high prevalence rate consisted of those <3 years old (19.05%) with no statistically significant difference (p = 0.821).

Influence of bovine trypanosomiasis on hematocrit: Of the 96 animals with hematocrit less than or equal to 24, only 11 (11.46%) had bovine trypanosomiasis. In the dry season, 20.93% (9/43) of trypanosomiasis-positive animals had haematocrit lower or equal to 24 with no statistically significant difference (p = 0.690), while in the rainy season only 3.92% (2/51) of positive animals had haematocrit less than or equal to 24 with no statistically significant difference (p = 0.530) in Table 4.

Influence of bovine trypanosomiasis on the body condition score: Based on the body condition score (BCS) of the animals, only 21.28% (20/94) of trypanosomiasis-positive cases were identified to exhibit a poor body condition. These 20 (10.99%) trypanosomiasis-positive poor condition animals were inferior to animals with medium and good body conditions and infected with trypanosomes (74: 13.28%). This difference was not statistically significant (p = 0.420). In the dry season in Table 5, the medium BCS animals were the most infected (12.5%) compared to poor BCS animals (2.5%) and this difference was significant (p = 0.028). In the rainy season, the most infected animals were those with a poor (17.65%) BCS while those with medium (14.61%) BCS were the least infected with a non-statistically significant difference (p = 0.782) (Table 5).

DISCUSSION

The present study that was conducted at the Ngaoundere Municipal Abattoir confirmed the presence of trypanosomiasis in cattle with a parasitological prevalence of 12.72%. This result was superior to that reported by Mamoudou et al.,¹¹ in Mayo-Rey with prevalence rates of 11.5 and 9%, respectively. This difference may be the animals slaughtered at the Ngaoundere Municipal Abattoir originated from bovine trypanosomiasis endemic areas such as Mayo-Rey with the highest number of cattle recorded in the Ngaoundere Abattoir. Conversely, this result was less than 29.4%, obtained in Almé in the Faro and Déo division⁹. This difference may be due to the nature of study areas in terms of tsetse fly infestation, where Almé is a tsetse infested area compared to Vina with continuous and intensive ongoing vector control. It was interesting to know that those animals coming from the Mayo-Rey Division were more highly infected with trypanosomiasis than those from the Vina Division. This finding was not surprising because Mayo-Rey-harbours dense tsetse and tabanids pockets as already reported that could be implicated in transmission as compared to Vina division with no reports on tsetse^1,2,11,15. Regarding season, the prevalence is significantly higher in the rainy season (15.60%) compared to the dry season (10.44%), probably due to the highest tsetse and other fly vectors densities occurring during this season in the Plateau of Adamawa¹⁶. This increase in prevalence during the rainy season corroborates the results obtained in Mayo-Rey¹⁰. In the present study, Trypanosoma congolense was the predominant species. The predominance of this species has been previously reported in Ngaoundere¹⁷. This indicates that there is contact between the host animals and tsetse flies and other biting dipterous insects that could lead to high transmission in those areas¹¹. The very low parasitaemia observed in this present study was similar to that reported by Mpouam et al.¹⁸. This result reflects the regular use of trypanocides by farmers in their breeding areas. The highest parasitaemia levels (5×10³ to 5×10⁴) were observed in infections caused by Trypanosoma congolense. This observation was consistent with that of Mpouam et al.¹⁸ and could be explained by the fact that Trypanosoma congolense is the most pathogenic species for cattle.

The average hematocrit of the animals was 30.09 and did not differ from that (30.2%) obtained in Vina¹⁸. The proportion of individuals with hematocrit less than 24 was higher in the dry season (18.69%) than in the rainy season (5.81%). This increase in mean hematocrit from the dry season to the rainy season was also recorded by Mamoudou et al.¹¹. This may be as result of increased pasture availability associated with increased nutrient values during the rainy season. No endogenous factor (sex, age and breed) considered had a significant influence on the prevalence of bovine trypanosomiasis at the Ngaoundere Municipal Abattoir and this observation was similar to that made by other authors^9,19.

The mean hematocrit of positive animals for trypanosomiasis was lower than that of negative animals. This decrease in mean hematocrit in positive animals was significant during the dry season. Several authors have reported a decrease in mean hematocrit in animals infected with trypanosomiasis^10,11. This result can be explained by the fact that bovine trypanosomiasis causes a sharp decrease in the number of red blood cells, resulting in a drop in the hematocrit. The scarcity of pasture and stress related to the search for food during the dry season, this has contributed to the poor body condition of the animals during this period. The influence of trypanosome species and degree of parasitaemia were not significant in trypanosomiasis-positive animals. However, mean hematocrit was lower in cattle infected with Trypanosoma congolense and especially in cattle with mixed infections due to Trypanosoma congolense + Trypanosoma vivax and Trypanosoma congolense + Trypanosoma brucei. These results were similar to those obtained by Mamoudou et al.¹³ and could be explained by the fact that Trypanosoma congolense is the most pathogenic species of cattle and its presence in the host system greatly reduces its red blood cell count. Only 11.46% of animals with hematocrit less than or equal to 24 had trypanosomiasis, hence the existence of other factors or anaemia-causing diseases such as gastrointestinal helminthiasis²⁰, haemoparasites, ectoparasites (ticks)²¹ and nutritional deficiencies¹¹ cannot be rolled-out. Several studies have shown that animals parasitized by both trypanosomes and helminths have lower hematocrit compared to animals with a single infection²⁰.

Of a total of 182 poor BCS animals, only 20 were trypanosomiasis positive. These 20 trypanosomiasis-positive poor BCS animals were inferior to animals with medium and good BCS infected with trypanosome (74). This result was different from that obtained by Samdi et al.²² in a study conducted at the Kaduna Abattoir in Nigeria. The decrease in the BCS could be partly linked to Trypanosoma spp., infection as well as other factors including gastrointestinal helminthiasis, ectoparasitosis, haemoparasites is transmitted by ticks, stress and nutritional deficiencies during the dry season¹¹.

CONCLUSION

From the abattoir survey, the parasitological prevalence was 12.72%. This prevalence was significantly higher in the rainy season than in the dry season and Trypanosoma congolense was the most frequent species. Infected cattle had lower hematocrit values than uninfected cattle. Poor body condition animals were highly infected with this parasite. This study confirmed the presence of trypanosomiasis in cattle slaughtered in Ngaoundere and indicates that a high risk could be from cattle coming from the tsetse infested Mayo-Rey Division in the North Region.

SIGNIFICANCE STATEMENT

Three pathogenic Trypanosoma spp. and their mixed infections occurred in cattle slaughtered at the Ngaoundere Municipal Abattoir. High cases of bovine trypanosomiasis were identified in cattle originating from the tsetse infested Mayo-Rey Division in the North Region which could be a possible source of introduction of the disease in the tsetse free Ngaoundere of Cameroon.