HAEMATOLOGICAL CHANGES IN HORSES EXPERIMENTALLY INFECTED WITH TRYPANOSOMA EVANSI.
MODIFICTIONS HEMOTOLOGIQUES CHEZ LES CHEVAUX INFECTES EXPERIMENTALEMENT PAR TRYPANOSOMA EVANSI.
Raymond E. Mdachi1, John M. Kagira11, Grace A. Murilla1 & Frans van Gool2
1Kenya Agricultural Research Institute-Trypanosomiasis Research Centre
P.O. Box 352 Kikuyu, Kenya.
2Merial SAS, 29Avenue Tony Garnier BP 7123,69348 Lyon Cedex 07 Lyon, France
Une enquête a été menée pour déterminer les paramètres hématologiques chez des chevaux lors d’une infection expérimentale par Trypanosoma evansi. Deux groupes de chevaux ont été infectés par voie intraveineuse avec 104 de trypanosomes de l’isolat de Trypanosoma evansi (KETRI 2426). Dans le premier groupe (infection aiguë), les animaux étaient traités à la mélarsomine cystéamine à raison de 0,25 mg/kg au bout de 4 jours d’infection, et dans le second groupe (infection chronique), les animaux étaient traités avec le même produit après 30 jours d’infection.
Qu’elle soit aiguë ou chronique, l’infection a causé des baisses considérables (p<0,001) du nombre d’erythrocytes et des valeurs de l’ hématocrite et de l’hémoglobine. De même, la numération de thrombocytes a baissé pendant les infections aiguë (p<0,01) et chronique (p<0,0001). Par contre, le nombre de leucocytes a beaucoup diminué (p<0,01) pendant l’infection aiguë, alors qu’il s’est considérablement accru (p<0,0001) pendant l’infection chronique. Les autres indices érythrocytaires (le volume corpusculaire moyen, l’hémoglobine corpusculaire moyenne et la concentration corpusculaire moyenne de l’hémoglobine) ont considérablement baissé (p<0,001) pendant l’infection chronique.
Le traitement des chevaux ayant une infection aiguë a entrainé, au bout de 48 heures, une réaction et un rétablissement des niveaux pré-infection de l’érythrocyte, du thrombocyte et du leucocyte. Mais chez les chevaux ayant une infection chronique, les indices érythrocytaires et les leucocytes ont changé 7 à 9 jours après le traitement, et les valeurs pré-infection n’ont pas été recouvrées pendant les 60 jours de suivi. Cela montre qu’il est nécessaire de procéder à un suivi post-traitement plus rigoureux pendant plus de 60 jours chez les chevaux souffrant d’une infection chronique sur le terrain.
Haematological parameter changes were investigated in horses during the course of an experimental Trypanosoma evansi infection. Two groups of horses were infected intravenously with 104 trypanosomes of the Trypanosoma evansi (KETRI 2426) isolate. In one group (acute), the animals were treated with melarsomine cysteamine at 0.25 mg/kg after 4 days of infection and in the other group (chronic) the animals were treated after 30 days of infection with the same drug. Erythrocyte indices, platelet and leucocyte counts were monitored three times a week before, during and after treatment for 60 days.
Both acute and chronic infections caused significant (p<0.001) decreases in erythrocyte, count, haematocrit and haemoglobin values. Similarly, platelet counts decreased during the acute (p<0.01) and chronic (p<0.0001) infections. In contrast, the leucocytes decreased significantly (p<0.01) during the acute infection whereas during the chronic infection the leucocytes increased significantly (p<0.0001). The other erythrocyte indices (MCV, MCH and MCHC) did not vary (p>0.05), during the acute infection, whereas these indices decreased significantly (p<0.001) during the chronic infection, an indication of development of normocytic normochromic and microcytic hypochromic anaemia respectively.
Treatment of the horses with the acute infection elicited response and recovery of erythrocyte, platelet and leucocytes to pre-infection level after 48 hrs in all horses. However, in the chronic infection the erythrocyte indices and leucocytes responded after 7-9 days after treatment and pre-infection values were not attained in the 60 days of follow up. In addition, the response of haematological changes was significantly different between horses. This suggests that closer post treatment monitoring for a longer period than 60 days of chronic infected horses in the field is necessary.
Surracaused by Trypanosoma evansi and transmitted by haematophagous flies is the most widely distributed of the pathogenic animal trypanosomiases. Its spread into South East Asia has occurred only relatively recently, and the serious epidemics of surra that were recorded in the early years of the 20th century in Indonesia and the Philippines, suggest that it could have spread into these regions within the last 100 years (Luckins, 1988; Lun et al. 1993). Similar outbreaks of disease occurred also in South America in the 19th century. In Africa, T. evansi occurs in areas beyond the northernmost limits of the tsetse fly and affects livestock in Kenya, Somalia, Ethiopia, Eritrea and Sudan. Countries in North Africa are also affected.
Trypanosoma evansi affects domesticated livestock in Asia, Africa and Central and South America. Surra is the principal protozoal disease of horses and wildlife in South America causing several hundred deaths of horses and other species every year. Several outbreaks have caused a high mortality in horses, of between 50.5% -75% (Silva et al., 1995a). In horses, the clinical and haematological signs observed include progressive anaemia, edema of the legs and lower parts, fever, lethargy, loss of appetite (Silva et al., 1995b; Mdachi et al unpublished data), weakness, lacrimation, abortion and loss of condition (Silva et al., 1995c).
Control of surra is mainly through use of trypanocidal drugs. Among the available drugs currently being used Melarsenoxide cysteamine (Cymelarsan®) is the most affective especially at late stage infection where relapses are most likely to occur (Naomi et al 2002) or in animals that exhibit nervous signs (Mdachi et al unpublished).
To investigate the nature and occurrence of anaemia in acute and chronic infections in horses, a study was conducted where haematological changes were monitored in horses experimentally infected with Trypanosoma evansi and treated with a curative drug at the acute and chronic stage of infection.
Materials and methods
Nine trypanosome naïve ponies purchased from a tsetse free area in Nairobi district were used. The horses were kept in a fly proof accommodation and allowed to acclimatized for three weeks. During this period the horses were de-wormed and sprayed for any ectoparasites. The horses were maintained on a ration of good quality hay supplemented with commercial horse pellets, proteins and mineral salts and water ad libitum.
Trypanosoma evansi isolate used was KETRI 2426 isolated from the field and sensitive to Cymelarsan® in mice at 0.5 mg/kg bodyweight. The horses were infected with Trypanosoma evansi (KETRI 2624) intravenously with 104 trypanosomes each and were bled 3 times a week from the Jugular vein into heparinised vacutainer tubes. Pre-infection samples were collected for 14 days before the animals were experimentally infected. Haematological parameters were determined using an automated haematology analyser (Coulter Ac.T diff, Beckman coulter). The parameters evaluated were total red blood cell (RBC) counts, haemoglobin (HB), haematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), total platelet count and total white blood cell (WBC) count.
Six of the nine horses were treated with Melarsenoxide cysteamine (Cymelarsan®) at 0.25 mg/kg bw on day four of infection (on set of parasitaemia) and the remaining three were treated on day 30 of infection at the same dose rate. The animals were followed for 60 days after treatment. Regression analysis of the hematological parameter was carried out to determine their changes with time post infection and treatment. Analysis of variance (ANOVA) was used to determine differences in haematological parameters of horses with acute and chronic infections.
During the 14 days pre infection period there were no significant changes (p>0.05) in RBC, HB, HCT, MCV, MCH, MCHC, and WBC values in the horses. However, platelet counts increased significantly (r2 = 0.546; slope=8.69; p<0.01) within the two weeks.
In horses with the chronic infection, there were no significant (p>0.05) differences in the haematological parameters between horses before and during infection. However, after treatment, the haematological parameters varied significantly (p<0.001) between animals.
In the acute infection, there were significant (p<0.001) differences in the haematological parameters between horses before infection and after treatment respectively. However during the four days of infection, the haematological parameters did not vary significantly (p>0.05) between horses.
In the acute infection, the total RBC counts, HCT and HB values dropped significantly (p<0.001) from pre infection mean values of (9.05 ± 0.15) x106/ul, 40.7 ± 0.60% and 13.4±0.20 g/dl to minimum mean values of (7.90 ± 0.29) x106/ul, 35.3 ± 0.96% and 11.5±0.31 g/dl respectively within three days after infection. Following treatment, the RBC values increased significantly (p<0.05) to mean values of (9.10 ± 0.37) x106/ul on day five after treatment, whereas the increase in HCT and HGB values to mean values of 37.2 ± 1.74% and 13.1 ± 0.59 g/dl on day five after treatment was not significant (p>0.05). Thereafter the three parameters did not change significantly (p<0.05) with days post treatment to the end of the experimental period.
During the chronic infection, the mean total RBC counts, HCT and HB values dropped significantly (p<0.001) from pre infection values of (10.7 ± 0.16) x106/ul, 51.4 ± 0.77% and 17.2±0.27 g/dl to minimum values of (8.79 ± 0.62) x106/ul, 36.9 ± 0.82% and 13.3±0.33 g/dl respectively. Following treatment on day 30 after infection, RBC, HCT and HGB values increased significantly (p<0.001) from day 7 after treatment to maximum mean values of (10.2 ± 0.50) x106/ul, 47.5 ± 2.12 % and 16.4±0.65 g/dl respectively on day 21 after treatment. Thereafter, the values of the three parameters decreased to low values of (7.98 ± 0.52) x106/ul, 38.0 ± 1.95 % and 12.9±0.67 g/dl respectively by the end of the experimental period.
In the acute infection, the MCV values (45.0 ± 2.04 fl) after infection did not vary significantly (p<0.05) from pre-infection values (45.3 ± 1.0 fl). Following treatment on day 4 of infection, the MCV values did not vary significantly (p>0.05) from pre-treatment values (45.0 ± 2.04 fl) by the end of the experimental period (42.2 ± 1.61 fl). In the chronic infection, the MCV values in horses declined significantly (p<0.01) during infection from pre-infection mean value of 47.9 ± 0.31fl to minimum mean values of 41.5±0.52fl. Following treatment, MCV values increased significantly (p<0.01) to maximum mean value of 47.7 ± 0.64fl then leveled off to a mean value of 47.3±0.20fl (p>0.05) by the end of the experimental period.
In the acute infection, the MCH and MCHC values (14.7±0.65 pg and 32.7 ± 0.07% respectively) following infection did not vary significantly (p<0.05) from pre-infection values (14.9±0.33 pg and 33.0 ± 0.08 % respectively). Following treatment on day 4 of infection, MCH and MCHC values (14.5±0.53 pg and 34.4±0.16 % respectively) by the end of the experimental period did vary significantly (p>0.05) from pre-treatment values (14.6±0.62 pg and 35.3 ± 0.21% respectively). In the chronic infection, the MCH values decline from pre infection mean values of 16.1 ± 0.11 pg to 15.3 ± 0.15 by 30 days after infection was not significant (p=0.058). However, the MCHC increased significantly (p<0.05) during infection from pre-infection mean values of 33.6 ± 0.14 to 36.3±0.18 by 30 days after infection. Following treatment, MCH and MCHC values increased significantly to 16.5±0.27 pg (p<0.001) and 34.7±0.20 (p=0.034) by the end of the experimental period.
In the acute infection, the platelet counts decreased from pre-infection mean values of (97.0±8.48) x 103/ul to significantly (p<0.05) lower values of (68.2±11.4) x 103/ul within 3 days of infection (Figure 5). Following treatment on day 4 after infection, platelet counts increased to significantly (p<0.05) higher mean values of (159 ± 17.1) x103/ul on day five after treatment. Thereafter, platelet counts decreased significantly (p<0.001) to mean values of (104 ± 19.4) x103/ul by the end of the experimental period. In the chronic infection, the total platelet counts decreased significantly (p<0.001) from pre-infection mean values of (150±32.4) x 103/ul to a minimum of (25.5±7.76) x 103/ul on day 30 after treatment. Following treatment, the values rose significantly (p<0.001) to a maximum mean value of (260 ±111) x 103/ul on day 18 after treatment. Thereafter the counts decreased to a mean value of (138±9.82) by the end of the experimental period.
In the acute infection, the WBC counts decreased from pre-infection mean values of (7.74±0.21) x 103/ul to significantly (p<0.01) lower values of (6.85±0.45) x 103/ul within 3 days of infection. Following treatment on day 4 after infection, white cell counts increased to significantly (p<0.01) higher values of (8.63 ± 0.43) x103/ul on day five after treatment. Thereafter, WBC counts decreased significantly (p<0.001) to mean values of (7.15 ± 0.53) x103/ul by the end of the experimental period. In the chronic infection, WBC counts decreased from pre-infection values of (6.84±0.14) x 103/ul to a minimum of (5.40±0.21) x 103/ul on day 5 after infection and then increased significantly (p<0.001) to maximum values of (10.1±0.46) x 106/ul. Following treatment, the WBC counts decreased significantly (p<0.001) from day 9 after treatment to mean values of (6.03±0.12) x 106/ul at the end of the experimental period.
Experimental infections in horses with T. evansi are very rarely related to in literature and lack of information is observed. Most of studies published concern natural infections in horses that are mainly at the chronic stage. Anaemia is one of the most consistent finding in animal trypanosomosis and its nature is difficult to elucidate. In this study the erythrocyte indices that would assist in determining the type of aneamia were measured in the acute and chronic stage of T. evansi infections.
In the acute infection, the type of aneamia that was developing was normocytic normochromic, which was characterised by significant decrease of haemoglobin and unchanged mean corpuscular volumes and mean corpuscular haemoglobin values within the four days of infection. Treatment with Cymelarsan® at day 4 of infection stopped further development of anemia and there was recovery of erythrocyte indices to pre-infection values. In contrast, the type of anemia that was developing in the chronic infection in horses was microcytic hypochromic, characterised by significant decrease of haemoglobin and the other erythrocyte indices. The subsequent recovery following treatment delayed for more than a week.
Development of thrombocytopoenia and leucopoenia was observed within three days of the acute infection. This was characterised by significant decrease in platelet and leucocytes respectively. Treatment of the horses led to recovery of platelets and leucocytes and induction of transient thrombocytosis and leucocytosis for five days post treatment characterised by significant increase in platelets and leukocytes to levels above pre-infection values and subsequent decline thereafter to normal values by end of study period. Thrombocytopoenia was similarly observed in the chronic infected horses. However, the level was higher characterised by a six fold decrease of platelets. The degree of drug-induced thrombocytosis was also higher in chronic infected horses and took a longer period for the effect to disappear. Leucocytosis was observed in chronic infected horses, which persisted for more than a week after treatment. This is an indication that transient leucocytosis observed in acute infected horses was probably drug induced. However, this cannot be corroborated since there is very little information of effect of Cymelarsen in horses. It would be important to carry out studies on effect of the drug on haematological parameters of non-infected horses.
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