The efficacy of a mixture of trimethoprim and sulphaquinoxaline against Plasmodium gallinaceum malaria in the domesticated fowl Gallus gallus

Veterinary Parasitology 129 (2005) 193–207 www.elsevier.com/locate/vetpar

The efficacy of a mixture of trimethoprim and sulphaquinoxaline against Plasmodium gallinaceum malaria in the domesticated fowl Gallus gallus R.B. Williams Wellcome Research Laboratories, Berkhamsted, Hertfordshire, UK Received 16 August 2004; received in revised form 29 December 2004; accepted 8 January 2005

Abstract The apicomplexan parasite Plasmodium gallinaceum has not been much studied from the veterinary standpoint. Although it causes malaria in domesticated chickens, no effective drugs appear to be commercially available. A mixture of trimethoprim and sulphaquinoxaline (TMP/SQX, ratio 1:3), with a wide spectrum of activity against bacteria and coccidia, is here shown to be also efficacious against blood-induced P. gallinaceum malaria when administered therapeutically in the feed of chickens for 5-day periods, beginning on the day before infection, or on the day of infection, or up to four days after infection. Chickens were protected against mortality and reduction of weight gain. Three other criteria of efficacy, which showed good correlation with each other and also with the two commercial performance criteria, were the production of green diarrhoea (due to biliverdin), parasitaemia and reduced haematocrit values. When TMP/SQX treatments were initiated sooner than five days after infection, parasites were almost entirely eliminated from the blood, whereas treatments initiated later than four days after infection failed to protect birds against clinical disease. Birds protected by TMP/SQX against primary infection with P. gallinaceum were immune to clinical malaria when exposed to a severe blood-induced challenge of P. gallinaceum 28 days later. # 2005 Elsevier B.V. All rights reserved. Keywords: Chemotherapy; Chickens; Malaria; Plasmodium gallinaceum; Sulphaquinoxaline; Trimethoprim

1. Introduction Plasmodium gallinaceum, a malarial parasite (Apicomplexa: Haemospororida) of birds of the genus Gallus, was first described by Brumpt in 1935. Most work on P. gallinaceum has been medically orientated (Garnham, 1966), and its veterinary importance has been rather neglected (Williams, 2005). However, it E-mail address: [email protected].

can cause serious malaria in domesticated chickens (Gallus gallus) throughout southern Asia. It has been recorded from commercial and back-yard chickens in Nhatrang (Brumpt, 1935), the Philippines (Africa et al., 1940), Sri Lanka (Crawford, 1945), India (Rao et al., 1951; Das et al., 1952), Indonesia (Kraneveld and Mansjoer, 1953) and Malaysia (Omar and Ismail, 1962; Omar and Lim, 1962; Omar, 1968). That the parasite is endemic to Asia is strongly suggested by its discovery there in various wild jungle fowl of the

0304-4017/$ – see front matter # 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2005.01.011

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R.B. Williams / Veterinary Parasitology 129 (2005) 193–207

genus Gallus (Shortt et al., 1941; Fernando and Dissanaike, 1975; Amin-Babjee et al., 1985). Garnham (1966) discussed several reports of P. gallinaceum from outside of Asia, but concluded that all involved experimentally infected birds or doubtful identifications. Nevertheless, it has more recently been reported from free-range chickens in Tanzania (Permin, 1998) and Zimbabwe (Permin et al., 2002), perhaps having been introduced somehow in imported fowl (A. Permin, personal communication). This apparent expansion of the known geographical range of P. gallinaceum parallels that of P. juxtanucleare, which was for some time believed to occur only in the New World (Garnham, 1966) but has now been recorded from Asia (Garnham, 1966; AminBabjee et al., 1985) and Africa (Permin, 1998; Permin et al., 2002). In view of the extreme susceptibility of domesticated chickens to P. gallinaceum (e.g., Permin and Juhl, 2002; Williams, 2005) and the likelihood of infections being transmitted to them from wild jungle fowl by mosquitoes, it is important to assess the efficacy of potential avian antimalarial drugs for the protection of commercial and family flocks of poultry. According to Bermudez (2003), ‘‘Although avian models have been used extensively in chemotherapeutic studies, information on potential preventative medications or treatments is limited.’’ In fact, no avian antimalarial drugs seem to be currently commercially available. Some drugs are known to be effective against P. juxtanucleare (e.g., Shanta and Wan, 1975, 1976a, 1976b; Shanta et al., 1977, 1978), but no similar veterinary work appears to have been carried out hitherto with P. gallinaceum. Therefore, studies of the efficacy of a mixture of trimethoprim and sulphaquinoxaline against P. gallinaceum are now reported.

2. Materials and methods 2.1. Infection of birds The parasite used in this study was P. gallinaceum Brumpt, 1935 (strain 8A; Wellcome line; Berkhamsted subline), described by Williams (2005). Experimental malarial infections were induced by the injection into a jugular vein of each bird of one million

parasitized erythrocytes (from donor birds) suspended in 0.1 ml of physiological saline. The day of infection in each experiment was designated as day 0. 2.2. Experimental animals and husbandry The chickens (Gallus gallus) used were males of the Ross Brown egg-laying hybrid, 14 days of age, housed in wire-floored cages at 24 8C and 37% relative humidity with 23 h lighting. Groups of infected birds (IUC = infected, unmedicated controls) were maintained alongside healthy uninfected groups (UUC = uninfected, unmedicated controls). Birds were caged in groups of five and had free access to unmedicated drinking-water and the experimental chicken mash (LD5) described by Williams (1996). The diet of control birds (UUC and IUC) was unmedicated throughout each experiment, and that of infected, treated birds contained a mixture of trimethoprim (TMP) and sulphaquinoxaline (SQX) for periods of five days as described in Section 2.3. Bird welfare was monitored at least four times daily. All work was carried out in compliance with the contemporary UK legislation on animal welfare and experimentation; any birds observed to be in extremis were killed humanely. 2.3. Medication The drug formulation tested was ‘‘Tribrissen-SQX Poultry Formula’’ (ex Wellcome Foundation Ltd.), a soluble powder containing 16.5% w/w TMP and 53.65% w/w SQX sodium, to give a ratio of one part of TMP to three parts of SQX. The formulation was mixed into chicken mash at 500 g per tonne, to give a concentration of 332.5 ppm of total active ingredients. The medicated feed was offered to birds for periods of five days only, beginning at various times before or after infection (see Section 2.4). Periods of administration of medicated feed were described relative to the day of infection (e.g., days
1–3, 0–4, 1–5, etc.). 2.4. Experimental design Every treatment in each experiment included 15 birds (three groups of five). Every bird, except those in the UUC groups, was infected on day 0.

R.B. Williams / Veterinary Parasitology 129 (2005) 193–207

Experiment I comprised the following treatments: UUC, IUC, and six treatments of infected birds given medicated feed for five days, beginning one day before infection (i.e., during days
1–3 inclusive), on the day of infection (days 0–4 inclusive), or one (days 1–5 inclusive), two (days 2–6 inclusive), three (days 3–7 inclusive) or four days after infection (days 4–8 inclusive). Medicated birds received plain feed at all other times. On day 28, all birds except those in the UUC treatment were reinfected. As a same-age control (CUC = challenged, unmedicated controls), two cages of birds (=10) from the same batch of dayold chicks, maintained malaria-free alongside the previously described birds, received a primary infection. All birds were monitored for 12 days after the challenge infection (up to 40 days after the primary infection). Experiment II comprised UUC, IUC, and four treatments of medicated feed for five days (4–8, 5–9, 6–10 or 7–11 days after infection). Birds were monitored for 24 days after the primary infection. No challenge infections were administered. Measures of efficacy in each experiment were as follows: (1) Mortality: All birds that died were recorded, and the cause of death was established, throughout each experiment. (2) Green diarrhoea: The occurrence of typically malarious green diarrhoea under each cage of birds was assessed daily from day 4 until day 40 in experiment I, and until day 24 in experiment II. A subjective score of severity (0, 1, 2 or 3) was allocated for each cage. Results for each treatment are presented as means of the individual cage scores. (3) Parasitaemia: In experiment I, the parasitaemia of each bird in one random cage of each treatment was assessed daily from day 3 to day 28 inclusive, and on days 33, 34, 35, 39 and 40; and in experiment II, from day 3 to day 14 inclusive, and on days 17 to 21 and 24. The same cage originally selected from each treatment was monitored throughout. (4) Haematocrit: The erythrocyte haematocrit of each bird in one random cage (the same one as that monitored for parasitaemia) of each treatment was assessed daily from day 3 to day 28 inclusive in

195

experiment I; and from day 3 to day 14 inclusive in experiment II. (5) Bird body weight gain: Every bird was individually colour-marked and weighed on days 0, 7, 14, 21, 28, 35 and 40 in experiment I; or on days 0, 7 and 14 in experiment II. 2.5. Experimental procedures In general, methods followed those described by Williams (2005), where further details may be found. Each cage was equipped with a metal tray underneath, on which a sheet of paper was placed to receive the faeces; after scoring the green diarrhoea each day, the paper was replaced with a clean sheet. Parasitaemias were assessed by the examination of Giemsa-stained blood smears; the results are shown as percentages of erythrocytes infected, rounded off to the nearest integer. The procedure standardized by Williams (1986) was used for obtaining erythrocyte haematocrit values. 2.6. Statistical analyses Methods for statistical analysis are given with the experimental results where appropriate.

3. Results 3.1. Mortalities Mortalities due to malaria following a primary infection in both experiments are shown in Table 1. In the IUC groups in experiment I, 93.3% mortality occurred (Table 1). CUC birds in experiment I, which were 28 days older, suffered mortality of 40% after the challenge infection (Table 1); the lower mortality of these older birds is in accord with the age related resistance demonstrated by Williams (2005). The infected birds that received TMP/SQX for five days in experiment I, beginning the day before infection or up to four days after infection did not die after the primary infection. When the same previously infected and medicated birds received a challenge infection on the 28th day after the primary infection, none died during the further 12 days of monitoring, demonstrating an acquired protective immunity.

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R.B. Williams / Veterinary Parasitology 129 (2005) 193–207

Table 1 Mortalities of chickens due to malaria in experiments I and II after primary infection with P. gallinaceum (blood-induced) and during treatment with TMP/SQX; data for medicated birds after challenge in experiment I described in text Experiment no.

I I I I I I I I I II II II II II II

Infected

No Yes Yes Yes Yes Yes Yes Yes Yesa No Yes Yes Yes Yes Yes a

TMP/SQX treatment days

None (UUC) None (IUC)
1–3 0–4 1–5 2–6 3–7 4–8 None (CUC) None (UUC) None (IUC) 4–8 5–9 6–10 7–11

Number of dead on day shown following infection 5

6

7

8

9

10

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

0 13 0 0 0 0 0 0 4 0 3 0 2 4 6

0 1 0 0 0 0 0 0 0 0 4 0 0 1 6

0 0 0 0 0 0 0 0 0 0 0 0 0 1 0

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

0 0 0 0 0 0 0 0 0 0 1 0 0 0 0

Total dead

% Dead

Mean time to death (days)

0 14 0 0 0 0 0 0 4 0 8 0 2 6 12

0.0 93.3 0.0 0.0 0.0 0.0 0.0 0.0 40.0 0.0 53.3 0.0 13.3 40.0 80.0

– 6.1 – – – – – – 6.0 – 7.0 – 6.0 6.5 6.5

Challenge control, infected 28 days after primary infection in experiment I.

However, infected birds in experiment II, in which treatment was initiated on the fifth, sixth or seventh day after infection, suffered mortalities of 13.3%, 40% or 80%, respectively, during eight days following infection (Table 1). The longer treatment was delayed, the greater was the mortality, but there was no obvious effect on mean time to death (Table 1). Infected birds treated on days 4–8 did not die, confirming the result obtained in experiment I (Table 1). 3.2. Occurrence of green diarrhoea All chickens infected with P. gallinaceum, including those that were medicated, produced green diarrhoea during the development of disease. No green diarrhoea appeared before the fifth day after primary infection in either experiment (Tables 2 and 3). In experiment I, the initiation of 5-day TMP/SQX treatments of infected birds on the day before infection and up to two days after infection delayed the production of green faeces for 10–13 days (Table 2). Furthermore, the severity of the diarrhoea was much reduced compared with the associated IUC (Table 2). Treatments initiated three or four days after infection in experiment I did not delay production of green diarrhoea, but they reduced the initial period of patency to three or four days, which was followed by

normal faecal production for nine or 10 days, after which a second period of production of green diarrhoea occurred, though less severe than originally in the IUC (Table 2). In experiment II, TMP/SQX treatments initiated from four to seven days after infection either did not delay the first production of green diarrhoea, or delayed it for only one day (Table 2). Similarly to what was seen in experiment I, a period of eight to 10 days of normal faecal production followed the initial green faecal production; then a second production of green diarrhoea of reduced severity occurred (Table 2). The mortalities of birds treated beginning on days 5, 6 or 7 (Table 1) were associated with the first period of green faecal production in experiment II. Judging by the CUC, the challenge infection in experiment I, 28 days after the primary infection, apparently did not cause production of green diarrhoea in any previously infected birds, whether treated with TMP/SQX or not (Table 3). The green diarrhoea recorded in previously infected birds appeared rather to be a continuation of the effect of the primary infection. This is indicated by the fact that green diarrhoea occurred continuously from the production following primary infection (Table 2), through the day of challenge, and up to and beyond the beginning of production in the CUC (Table 3). Furthermore, the

0 2 1.7 1 1.3 1.3 1 1 0 – – – – –

26

0 0 1.7 1.3 1.7 1.7 1.3 2 0 – – – – – 0 1 1.7 1.7 2 2 2 2.7 0 – 0 3 3 1

0 0 1.3 1.7 2 1.7 1.7 2.3 0 – – – – –

24

0 2 2 2.3 3 2.7 2.3 3 0 – 0 2 2 2 0 2 2 2.3 3 2.7 3 3 0 – 1 2 3 1 0 2 2 3 3 3 3 2.7 0 – 1 1 3 1 (
) Not recorded.

21 20

0 3 2.7 2.7 2.7 3 3 2.7 0 – 1 3 3 2 0 3 3 3 2 3 3 1.7 0 – 1 2 3 1

19 18

0 3 2.7 2 0.7 2 1.7 1 0 – 2 0 0 0 0 3 2.3 1 0 0 0 0 0 – 0 0 0 0

17 16

0 3 1.3 0 0 0 0 0 0 – 0 0 0 0 0 3 0.3 0 0 0 0 0 0 – 0 0 0 0

15 14

0 3 0 0 0 0 0 0 0 1 0 0 0 0 0 3 0 0 0 0 0 0 0 1 0 0 0 0

13 12

0 3 0 0 0 0 0 0 0 1 0 0 0 0 0 3 0 0 0 0 0 0 0 2 0 0 0 0

11 10

0 3 0 0 0 0 0 0 0 2.5 0 0 0 1 0 3 0 0 0 0 0 0 0 3 0.3 0 2.3 3

9 8 7

0 3 0 0 0 0 0.3 0.7 0 3 1 2.3 2.7 3

6 5

0 1.7 0 0 0 0 1.3 2.3 0 0 1 0 0 0.7 No Yes Yes Yes Yes Yes Yes Yes No Yes Yes Yes Yes Yes I I I I I I I I II II II II II II

The normal range of erythrocyte haematocrit values in chickens of the same sex, age and breed as those used here is from 25% to 32% (Williams, 1986). Nevertheless, 5% of single haematocrit readings from healthy chickens might be expected to fall outside that range. In fact, 3.1% of the readings from the UUC birds in experiment I fell outside the normal range (Table 7), as did 3.3% of the readings from the UUC birds in experiment II (Table 8). In the present study, low values of 24% were regarded as indicative of a severe malarial infection.

Infected

3.4. Haematocrit values

None (UUC) None (IUC)
1–3 0–4 1–5 2–6 3–7 4–8 None (UUC) None (IUC) 4–8 5–9 6–10 7–11

0 3 0 0 0 0 1.3 1.7 0 2 0 2.3 2 2

0 3 0 0 0 0 0 0.3 0 3 0.7 1 3 3

22

23

25

0 2 1.7 1.3 1.3 1.7 1 1 0 – – – – –

severity of the challenge effect in the CUC was greater than in all of the previously infected birds, whether medicated or not, and it continued maximally while diarrhoea scores in the previously infected birds fell to zero, or nearly so (Table 3).

Tables 4–6 show the percentages of parasitaemia in chickens during development of primary and challenge infections. In experiment I, initiation of TMP/ SQX treatments on the day before primary infection, on the day of infection, or on the day after, delayed onset of parasitaemia by 10–13 days (Table 4). Treatments begun two to four days after infection did not delay onset, but the initial patent period was restricted to two to seven days, followed by negative parasitaemias for five to 10 days, after which a second patent period of rather low intensity began and continued for about three to eleven days (Table 4). The challenge infection, 28 days after the primary infection in experiment I, produced the expected high parasitaemias in the same-age controls (CUC), but had a negligible effect in all the previously infected, unmedicated and medicated birds (Table 5). In experiment II, treatments initiated four to seven days after primary infection did not delay the onset of parasitaemia, and deaths that occurred (Table 1) were associated with the initial patent period coinciding with that of the IUC (Table 6). In survivors, negative parasitaemias were observed for about four to 10 days following the initial period of patency, after which a second patent parasitaemia of much reduced intensity ensued.

TMP/SQX treatment days

Green diarrhoea scores on days shown following infection (mean scores of 3 groups per treatment)

197

3.3. Parasitaemia

Experiment no.

Table 2 Green diarrhoea observed in experiments I and II after primary infection with P. gallinaceum (blood-induced) and during treatment with TMP/SQX

27

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R.B. Williams / Veterinary Parasitology 129 (2005) 193–207

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R.B. Williams / Veterinary Parasitology 129 (2005) 193–207

Table 3 Green diarrhoea observed in experiment I after challenge infection with P. gallinaceum (blood-induced) following treatment with TMP/SQX Challenge infection

Primary infection

TMP/SQX treatment days

Green diarrhoea scores on days shown following infection (mean scores of three groups per treatment) 28

29

30

31

32

33

34

35

36

37

38

39

40

Yes No Yes Yes Yes Yes Yes Yes Yes

No No Yes Yes Yes Yes Yes Yes Yes

None (CUC) None (UUC) None (IUC)
1–3 0–4 1–5 2–6 3–7 4–8

0 0 2 1.7 1 1.3 1.3 1 1

0 0 3 1.7 1.3 1.3 1.3 1 1

0 0 1 1.7 1.3 1.3 1 0.7 1.3

0 0 0 2 1.3 1 1 0.3 1.3

0.5 0 1 1.7 1.3 0.7 0.7 0.3 0.7

2 0 1 1.3 1.3 1.3 1 0.7 1

3 0 1 1.3 1.7 1.3 1 0.7 1

3 0 0 1 1.7 1.3 1 0.7 0.7

3 0 0 0.7 1.7 1 1.3 0.7 0.7

3 0 0 0.3 1 0.3 1 0 0

3 0 0 0.3 0.7 0.3 0.3 0 0

3 0 0 0 0.7 0.3 0.3 0 0

3 0 0 0 0.3 0.3 0 0 0

CUC: challenged unmedicated control (two groups of same-age birds).

Table 7 shows the erythrocyte haematocrit values of the same infected chickens as those in Table 4, plus five UUC birds, for the first 28 days of experiment I. Mean values for each monitored group are shown in Fig. 1, highlighting the onset of anaemia following each treatment. Significant falls in haematocrit values usually occurred, three or four days after infection, in IUC birds, most of which died of malaria. In TMP/ SQX-medicated birds, subnormal haematocrit values (indicated by bold figures) occurred mostly in two distinct clusters (Table 7). Birds medicated beginning on days
1 to +1 relative to the primary infection

(day 0) developed subnormal haematocrit values 14– 17 days after infection. This cluster was continuous with that of birds medicated beginning two to four days after infection, in which subnormal values began to occur up to 19 days after infection. However, those latter birds also exhibited an earlier, well-separated cluster of subnormal haematocrit values, mostly spanning four to seven days after infection (Table 7). Occasional subnormal values were scattered randomly outside the clusters, as would be expected. The first fall in haematocrit values in birds medicated beginning two to four days after infection

Fig. 1. Daily mean haematocrit values of groups of five birds treated for five days with TMP/SQX, beginning at various times relative to infection with P. gallinaceum, compared with UUC and IUC groups (experiment I; data derived from Table 7).

Table 4 Parasitaemias observed in experiment I after primary infection (day 0) with P. gallinaceum (blood-induced) and during treatment with TMP/SQX

20

21

22

23

24

25

26

27

28

6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40

– – – +ve – +ve +ve 0.5
ve
ve 7 +ve +ve
ve 1 4 4 0.5 78 8 15 0.5 0.5 0.5 +ve +ve 0.5
ve +ve
ve
ve +ve
ve 1
ve

– – –
ve – +ve +ve +ve
ve
ve 5
ve
ve
ve +ve 0.5 1 +ve 52 1 4 +ve
ve 0.5 +ve +ve +ve
ve +ve +ve
ve
ve
ve 1
ve

– – –
ve – +ve
ve +ve
ve
ve 2
ve
ve +ve
ve +ve +ve
ve 14 +ve +ve
ve
ve
ve
ve
ve
ve +ve +ve
ve
ve
ve +ve
ve +ve

– – –
ve –
ve +ve
ve
ve
ve +ve
ve
ve
ve +ve +ve
ve
ve 3
ve +ve
ve
ve
ve +ve
ve
ve +ve
ve
ve
ve +ve +ve +ve +ve

– – –
ve –
ve
ve
ve
ve
ve +ve
ve
ve
ve +ve
ve
ve
ve 0.5
ve +ve +ve
ve +ve +ve
ve
ve
ve
ve
ve
ve
ve
ve
ve
ve

– – –
ve –
ve
ve
ve
ve
ve +ve
ve
ve
ve +ve +ve
ve
ve +ve
ve +ve
ve
ve
ve +ve
ve
ve
ve +ve +ve
ve
ve
+ve +ve

– – – +ve –
ve
ve
ve
ve
ve
ve
ve
ve
ve
ve
ve +ve
ve +ve
ve +ve
ve
ve +ve +ve
ve
ve
ve
ve
ve
ve
ve

ve
ve

– – – +ve –
ve
ve
ve
ve
ve +ve
ve
ve
ve +ve
ve +ve
ve +ve
ve
ve
ve
ve
ve
ve
ve
ve
ve
ve
ve
ve
ve

ve
ve

– – –
ve –
ve
ve
ve
ve
ve
ve +ve
ve
ve
ve
ve +ve
ve
ve
ve
ve
ve
ve
ve
ve
ve
ve
ve
ve
ve
ve
ve

ve
ve

None (IUC) None (IUC) None (IUC) None (IUC) None (IUC)
1–3
1–3
1–3
1–3
1–3 0–4 0–4 0–4 0–4 0–4 1–5 1–5 1–5 1–5 1–5 2–6 2–6 2–6 2–6 2–6 3–7 3–7 3–7 3–7 3–7 4–8 4–8 4–8 4–8 4–8

2 3 4 2 2
ve
ve
ve
ve
ve
ve
ve +ve
ve
ve
ve
ve
ve +ve
ve +ve 0.5 +ve 0.5 0.5 0.5 +ve 0.5 0.5 1 +ve 0.5 +ve 1 +ve

11 12 10 9 11
ve
ve
ve
ve
ve
ve
ve
ve
ve
ve
ve
ve
ve +ve
ve +ve 0.5 0.5 0.5 0.5 11 7 5 8 11 6 9 6 6 7

76y 68y 57y 65 62y
ve
ve
ve
ve
ve
ve
ve
ve
ve
ve
ve
ve
ve
ve
ve +ve +ve
ve 0.5 +ve 6 6 4 7 6 3 8 3 3 5

– – – 87 –
ve
ve
ve
ve
ve
ve
ve
ve
ve
ve
ve
ve
ve
ve
ve
ve
ve
ve
ve
ve 4 2 0.5 1 1 0.5 5 0.5 +ve 1

– – – 25 –
ve
ve
ve
ve
ve
ve
ve
ve
ve
ve
ve
ve
ve
ve
ve
ve
ve
ve
ve
ve +ve
ve
ve
ve
ve +ve +ve
ve +ve +ve

– – – 35 –
ve
ve
ve
ve
ve
ve
ve
ve
ve
ve
ve
ve
ve
ve
ve
ve
ve
ve
ve
ve +ve
ve
ve
ve
ve +ve +ve
ve
ve
ve

– – – 73 –
ve
ve
ve
ve
ve
ve
ve
ve
ve
ve
ve
ve
ve
ve
ve
ve
ve
ve
ve
ve +ve
ve
ve
ve
ve
ve
ve
ve
ve
ve

– – – 48 –
ve
ve
ve
ve
ve
ve
ve
ve
ve
ve
ve
ve
ve
ve
ve
ve
ve
ve
ve
ve
ve
ve
ve
ve
ve
ve
ve
ve
ve +ve

– – – 32 –
ve
ve
ve
ve
ve
ve
ve
ve
ve
ve
ve
ve
ve
ve
ve
ve
ve
ve
ve
ve
ve
ve
ve
ve
ve
ve
ve
ve
ve
ve

– – – 30 –
ve
ve
ve
ve
ve
ve
ve
ve
ve
ve
ve
ve
ve
ve
ve
ve
ve
ve
ve
ve
ve
ve
ve
ve
ve
ve
ve
ve
ve
ve

– – – 38 – 0.5 0.5 +ve +ve +ve
ve
ve +ve
ve
ve
ve
ve
ve
ve
ve
ve
ve
ve
ve
ve
ve
ve
ve
ve
ve
ve
ve
ve
ve
ve

– – – 13 – 4 4 +ve +ve 0.5
ve +ve 0.5 +ve
ve
ve
ve
ve
ve
ve
ve
ve
ve +ve
ve
ve
ve
ve
ve
ve
ve
ve
ve
ve
ve

– – – 3 – 6 5 14 +ve 0.5
ve 2 0.5 +ve +ve +ve
ve
ve
ve
ve
ve +ve +ve +ve +ve 0.5 +ve +ve
ve
ve
ve
ve
ve
ve
ve

– – – 1 – 19 47 13 +ve +ve
ve +ve 0.5 1 0.5 +ve +ve 0.5 +ve +ve +ve +ve 0.5 0.5 0.5 1 +ve +ve 0.5 +ve
ve 0.5 +ve
ve 0.5

– – – +ve – 56 42 2 1 +ve
ve
ve 1 0.5 1 1 +ve 1 +ve +ve 2 0.5 1 1 4 1 1
ve 1 +ve +ve 1
ve
ve 0.5

– – – +ve – 25 3 3 2 +ve 2 +ve +ve 1 6 3 +ve 1 3 2 4 1 1 2 13 2 +ve
ve 0.5 +ve 0.5 11 +ve +ve 2

– – – +ve – 1 +ve 0.5 +ve
ve 11 +ve +ve +ve 7 10 1 0.5 39 9 16 0.5 7 1 3 0.5 +ve
ve 1 +ve 0.5 2
ve +ve +ve

R.B. Williams / Veterinary Parasitology 129 (2005) 193–207

Bird no. TMP/SQX Parasitaemias on days shown following primary infection (birds from one group per treatment) treatment days 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

(y) Bird died of malaria; (
) reading not taken.

199

200

R.B. Williams / Veterinary Parasitology 129 (2005) 193–207

Table 5 Parasitaemias observed in experiment I after challenge infection with P. gallinaceum (blood-induced) 28 days after primary infection (day 0) Bird no.

Infected

TMP/SQX treatment days

A B C D E 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40

No No No No No Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes

None (CUC) None (CUC) None (CUC) None (CUC) None (CUC) None (IUC) None (IUC) None (IUC) None (IUC) None (IUC)
1–3
1–3
1–3
1–3
1–3 0–4 0–4 0–4 0–4 0–4 1–5 1–5 1–5 1–5 1–5 2–6 2–6 2–6 2–6 2–6 3–7 3–7 3–7 3–7 3–7 4–8 4–8 4–8 4–8 4–8

Parasitaemias on days shown following primary infection (birds from one group per treatment) 28

33

34

35

39

40


ve
ve
ve
ve
ve – – –
ve –
ve
ve
ve
ve
ve
ve +ve
ve
ve
ve
ve +ve
ve
ve
ve
ve
ve
ve
ve
ve
ve
ve
ve
ve
ve
ve
ve –
ve
ve

27 36 21y 23 30 – – –
ve –
ve
ve
ve
ve
ve
ve +ve
ve
ve +ve
ve
ve
ve +ve
ve
ve
ve +ve
ve
ve
ve +ve
ve
ve
ve
ve
ve –
ve
ve

85 76
82 79 – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – –

27 35 – 36 48 – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – –


ve
ve – 18 +ve – – – +ve – +ve
ve
ve
ve
ve
ve
ve
ve +ve
ve
ve
ve
ve
ve
ve
ve
ve
ve
ve
ve
ve
ve
ve
ve
ve
ve
ve –
ve
ve


ve
ve – 7
ve – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – –

(y) Bird died of malaria; (
) reading not taken (in IUC group, bird dead before day 28).

in experiment I was delayed slightly in comparison with the IUC birds. Fig. 1 shows the steady haematocrit values of the UUC, the early reduction in the IUC, and how the mean onset of anaemia in each TMP/SQX-medicated group was delayed, directly associated with the delays in the initiation of the medication periods. Haematocrits were not measured

after the challenge infections were administered in experiment I. In experiment II (Table 8), subnormal haematocrit values formed a single cluster mainly between four and eight days after infection. The cluster comprised the IUC and all birds medicated beginning four to seven days after infection. This cluster of subnormal

R.B. Williams / Veterinary Parasitology 129 (2005) 193–207

201

Table 6 Parasitaemias observed in experiment II after primary infection (day 0) with P. gallinaceum (blood-induced) and during treatment with TMP/ SQX Bird no. TMP/SQX Parasitaemias on days shown following infection (birds from one group per treatment) treatment days 3 4 5 6 7 8 9 10 11 12 13 14 17 18 19

20

21

24

6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30

– – – – – +ve +ve +ve +ve 2 0.5 +ve – 1 +ve – – – 4
ve
ve – +ve – +ve

– – – – – +ve +ve +ve
ve +ve 1 3 – +ve +ve – – – 1 +ve
ve –
ve – +ve

– – – – –
ve
ve
ve +ve
ve +ve +ve –
ve +ve – – –
ve +ve
ve –
ve –
ve

None None None None None 4–8 4–8 4–8 4–8 4–8 5–9 5–9 5–9 5–9 5–9 6–10 6–10 6–10 6–10 6–10 7–11 7–11 7–11 7–11 7–11

(IUC) (IUC) (IUC) (IUC) (IUC)

5 5 +ve +ve +ve 2 +ve +ve +ve +ve +ve +ve 0.5 +ve +ve 1 2 1 +ve +ve +ve +ve +ve
ve 1

9 8 1 +ve 1 5 2 2 3 1 1 +ve 5 2 0.5 6 7 3 1 7 +ve +ve +ve 0.5 2

50y 60y 12 6 9 45 18 22 16 11 25 +ve 39y 20 9 35y 41y 35y 17 28 2 4 7 15 8

– – 59 57y 61y 14 13 12 2 5 3 +ve – 30 2 – – – 82 79 17 27y 51 49y 75

– – 77y – – 3 2 2 +ve +ve +ve
ve – 5 +ve – – – 40 12 62 – 75 – 78

– – – – –
ve
ve +ve
ve
ve
ve
ve – 0.5
ve – – – +ve +ve 14 – 33 – 25

– – – – –
ve
ve
ve
ve
ve
ve
ve –
ve
ve – – –
ve
ve 2 – +ve – 26

– – – – –
ve
ve
ve
ve
ve
ve
ve –
ve
ve – – –
ve
ve
ve –
ve – 1

– – – – –
ve
ve
ve
ve
ve
ve
ve –
ve
ve – – –
ve
ve
ve –
ve –
ve

– – – – –
ve
ve
ve
ve
ve
ve
ve –
ve
ve – – –
ve
ve
ve –
ve –
ve

– – – – – +ve
ve
ve
ve
ve
ve
ve –
ve
ve – – –
ve
ve
ve –
ve –
ve

– – – – –
ve
ve
ve
ve
ve
ve
ve –
ve
ve – – –
ve
ve
ve –
ve –
ve

– – – – – +ve 0.5 +ve +ve +ve 0.5
ve – 1
ve – – – +ve +ve 0.5 – +ve – +ve

– – – – – +ve +ve +ve
ve 2 3
ve – 12
ve – – – 4 1
ve – +ve – +ve

– – – – – +ve +ve 1
ve 5 5 +ve – 6 +ve – – – 15 +ve
ve – 11 – +ve

(y) Bird died of malaria; (
) reading not taken.

values coincided with the timing of the first cluster in birds medicated beginning two to four days after infection in experiment I (Table 7). 3.5. Weight gains In experiment I, all treatments of TMP/SQX protected infected birds against reduction of weight gain during the acute phase of malaria (days 0–7) (Table 9). During subsequent weekly periods of weight measurement (days 7–14, 14–21 and 21–28), some statistically significant differences occurred between mean weight gains due to various TMP/ SQX treatments, but no particular treatment consistently produced a worse performance than that of the UUC during every period (Table 9). The weight gain of the IUC during days 0–28 was only 58.7% of that of the UUC birds, but because it represented only a single surviving bird, it was excluded from the statistical analyses.

On the 28th day after the primary infection in experiment I, all surviving birds except those of the UUC were challenged with a second infection of the same magnitude as the first. Birds of the same age, maintained malaria-free under the same conditions, were used as a challenged unmedicated control (CUC). During the acute phase of the challenge infection (days 28–35), the mean weight gain of those birds in the CUC that survived the challenge was poorer than that of the UUC (P < 0.001) and of each of the TMP/SQX treatments (P < 0.001) (Table 9). During the recovery phase following the challenge infection, (days 35–40), the mean weight gain of the CUC birds was not statistically significantly different from that of the UUC birds. However, it was still statistically significantly lower than the mean weight gains of birds treated with TMP/SQX on days
1–3 and 0–4. During the challenge period of 28–40 days, the mean weight gains of all previously medicated birds, except for those treated on days 2–6, were

202

Table 7 Erythrocyte haematocrit values observed in experiment I after primary infection (day 0) with P. gallinaceum (blood-induced) and during treatment with TMP/SQX Infected

TMP/SQX treatment days

Haematocrits on days shown following infection (birds from one group per treatment) 3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40

No No No No No Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes

None (UUC) None (UUC) None (UUC) None (UUC) None (UUC) None (IUC) None (IUC) None (IUC) None (IUC) None (IUC)
1–3
1–3
1–3
1–3
1–3 0–4 0–4 0–4 0–4 0–4 1–5 1–5 1–5 1–5 1–5 2–6 2–6 2–6 2–6 2–6 3–7 3–7 3–7 3–7 3–7 4–8 4–8 4–8 4–8 4–8

27 27 29 26 26 25 23 25 26 27 25 29 26 25 27 27 26 26 26 27 25 28 26 25 26 27 25 24 25 28 28 24 25 26 26 27 27 28 25 25

28 27 29 27 25 22 20 22 23 28 25 26 26 27 27 27 25 27 24 24 27 27 26 25 24 27 24 22 22 28 26 23 24 27 22 26 27 25 24 23

27 27 29 27 25 21y 19y 19y 21 28y 24 30 28 29 29 28 26 29 25 25 26 29 26 26 26 25 23 22 21 27 26 22 22 23 24 23 21 23 20 21

29 28 29 28 25


19
27 28 26 30 27 28 28 27 23 25 26 27 26 26 25 26 27 22 24 27 25 22 20 24 23 23 25 23 21 24

29 27 27 27 26


19
32 31 27 32 30 30 29 30 26 26 27 28 27 27 24 28 28 25 26 29 26 24 24 23 25 25 22 26 26 23

29 29 30 29 28


21
28 28 25 31 28 28 28 28 26 27 27 28 26 27 26 27 29 27 27 30 30 29 26 26 29 28 27 29 28 26

29 29 30 28 26


22
27 29 26 30 27 27 27 27 26 25 29 27 26 26 25 28 29 25 27 28 31 29 29 27 29 29 29 29 30 26

28 28 30 28 26


25
28 30 26 31 28 29 28 28 28 25 28 26 27 27 25 30 30 24 26 27 32 31 29 29 30 32 29 30 29 28

30 28 30 26 26


22
29 28 27 29 26 30 30 28 28 26 28 28 28 28 25 30 30 26 27 30 33 32 32 29 30 33 30 30 30 27

28 29 29 26 26


24
28 30 27 30 27 29 29 27 27 25 28 28 28 27 26 29 32 25 31 29 34 33 32 32 31 30 29 31 29 27

29 28 28 27 26


24
28 29 27 30 27 30 28 27 26 26 28 30 28 27 25 29 31 24 30 30 34 32 30 30 31 33 30 30 29 28

31 27 28 28 26


22
24 27 22 26 26 29 29 25 26 26 29 27 27 27 25 31 30 26 29 28 33 33 32 29 29 32 33 30 27 26

28 27 25 26 25


25
21 24 23 23 21 29 29 22 25 25 27 28 28 30 25 30 29 24 29 28 31 27 28 28 27 34 29 31 27 26

28 26 29 26 25


26
17 17 19 24 19 28 26 17 22 23 26 26 25 25 25 26 27 24 27 25 29 30 27 30 28 31 29 30 26 27

29 27 29 25 24


26
15 14 15 24 22 30 24 17 19 20 25 24 22 26 22 27 26 23 23 25 23 28 20 24 24 32 30 31 26 25

29 26 29 26 25


25
14 18 16 22 24 26 26 20 15 14 23 20 19 24 22 24 19 14 15 19 18 18 16 17 17 30 25 30 26 24

29 27 28 26 27


24
19 25 17 18 22 25 25 22 21 14 16 20 17 21 16 19 22 16 14 16 20 16 21 20 21 20 13 23 19 14

29 27 29 27 26


23
20 27 20 25 23 16 23 22 22 15 14 18 20 12 11 20 22 16 18 20 22 21 21 21 20 16 18 25 18 18

32 28 30 27 25


26
25 27 22 28 22 15 23 23 23 20 20 14 21 11 12 19 22 19 20 26 21 21 20 22 19 20 22 24 20 21

29 27 28 27 24


27
24 26 22 30 25 17 21 22 20 21 25 17 24 17 21 27 23 23 22 24 24 20 20 21 19 22 22 22 19 21

29 28 29 26 25


27
24 25 20 30 24 20 23 23 22 21 26 24 26 20 22 25 23 22 22 24 24 21 19 21 22 25 23 22 26 21

29 25 27 27 26


30
21 25 18 28 25 26 25 22 22 19 24 26 25 21 24 27 23 22 23 23 25 21 17 22 24 24 24 21 28 20

29 27 27 26 25


30
25 23 17 30 27 27 26 22 26 19 26 26 25 21 24 26 23 22 24 22 27 22 19 23 24 25 26
27 21

29 24 25 26 24


32
23 26 24 26 26 28 26 25 27 24 26 23 26 23 23 30 25 22 23 24 26 24 24 26 23 26 28
30 22

27 25 25 26 25


27
22 25 25 22 26 26 26 25 27 26 24 24 28 19 20 30 24 21 20 23 27 26 24 27 25 29 29
29 22

28 25 27 25 26


24
26 27 27 23 26 27 24 25 25 26 26 22 24 18 22 28 25 22 21 23 28 25 25 28 24 27 29
28 23

Subnormal values are shown in bold. (y) Bird died of malaria; (
) reading not taken.

R.B. Williams / Veterinary Parasitology 129 (2005) 193–207

Bird no.

R.B. Williams / Veterinary Parasitology 129 (2005) 193–207

203

Table 8 Erythrocyte haematocrit values observed in experiment II after primary infection (day 0) with P. gallinaceum (blood-induced) and during treatment with TMP/SQX Bird no.

Infected

TMP/SQX treatment days

Haematocrits on days shown following infection (birds from one group per treatment) 3

4

5

6

7

8

9

10

11

12

13

14

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30

No No No No No Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes

None None None None None None None None None None 4–8 4–8 4–8 4–8 4–8 5–9 5–9 5–9 5–9 5–9 6–10 6–10 6–10 6–10 6–10 7–11 7–11 7–11 7–11 7–11

29 28 28 26 27 28 26 32 26 28 26 28 29 29 25 27 28 26 25 25 25 27 24 23 28 24 31 29 27 25

29 27 28 29 27 29 21 24 24 26 24 26 28 26 23 27 27 24 24 22 23 23 26 27 30 22 27 28 26 25

26 28 29 28 23 24y 22y 22 23 26 24 28 26 25 23 26 25 29y 23 25 25y 24y 22y 24 26 22 26 26 25 24

26 28 26 29 27 – – 22 27y 24y 22 23 21 24 22 25 20 – 22 21 – – – 23 23 20 26y 24 26y 21

31 27 30 29 28 – – 18y – – 20 20 24 25 19 22 23 – 18 24 – – – 23 17 15 – 24 – 15

31 28 30 29 27 – – – – – 26 24 25 26 19 26 25 – 20 24 – – – 17 23 15 – 16 – 13

32 29 30 29 27 – – – – – 26 28 27 30 25 26 25 – 26 27 – – – 29 30 20 – 21 – 15

33 29 32 29 28 – – – – – 30 29 35 33 26 28 27 – 26 28 – – – 27 29 22 – 30 – 23

32 29 31 29 27 – – – – – 29 30 30 28 29 29 27 – 29 29 – – – 31 32 29 – 30 – 27

29 27 31 29 29 – – – – – 30 28 31 31 29 30 27 – 29 27 – – – 31 29 27 – 31 – 30

30 28 32 28 28 – – – – – 29 29 30 29 28 31 29 – 29 28 – – – 33 31 32 – 33 – 28

30 27 29 29 29 – – – – – 27 29 30 30 29 32 30 – 29 29 – – – 34 29 30 – 31 – 26

(UUC) (UUC) (UUC) (UUC) (UUC) (IUC) (IUC) (IUC) (IUC) (IUC)

Subnormal values are shown in bold. (y) Bird died of malaria; (
) reading not taken.

between 96.9% and 104.3% of the UUC birds, showing that medication had not interfered with the acquisition of protective immunity. There was a tendency for the medication treatments begun the earliest to produce the greatest mean weight gains throughout the whole period, from the primary infection to recovery from the challenge infection (Table 9). In experiment II, in which later periods of medication were tested, that tendency was confirmed, with statistically significant differences during the acute phase of infection (days 0–7) between the mean weight gain of the UUC birds and those of the smallest weight gains of birds treated with TMP/SQX on days 6–10 and 7–11 (Table 10). The overall mean

weight gains of all TMP/SQX-treated birds as percentages of the UUC in experiment II were generally lower than those of the earlier initiated treatments in experiment I.

4. Discussion Although P. gallinaceum has been widely used as a tool for screening potential human antimalarial agents (Peters, 1974; Richards, 1984), it has not necessarily given results that are in accord with those obtained by using mammalian plasmodia such as P. berghei in mice (e.g., Ryley and Peters, 1970). However, the ideal model for identifying potential antimalarial

204 Table 9 Weight gains (S.D.), during 40 days, of chickens infected on day 0 with P. gallinaceum (blood-induced) at 14 days of age in experiment I, followed by a challenge at 28 days Mean weight gain (g) days 0–7a

Mean weight gain (g) days 7–14b

Mean weight gain (g) days 14–21c

Mean weight gain (g) days 21–28d

Mean weight gain (g) days 0–28e

Weight gain days 0–28 as % of UUC

Mean weight gain (g) days 28–35f

Mean weight gain (g) days 35–40g

Mean weight gain (g) days 28–40h

Weight gain days 28–40 as % of UUC

UUC IUCi CUCj TMP/SQX TMP/SQX TMP/SQX TMP/SQX TMP/SQX TMP/SQX

94.27  7.92 39  0 – 94.64  8.19 91.71  9.17 92.80  8.14 88.79  6.84 90.53  13.92 85.64  8.88

132.53  16.37 72  0 – 134.43  10.35 129.57  16.29 121.53  18.55 122.36  15.22 117.00  15.79 118.93  12.53

160.27  33.47 123  0 – 137.29  38.19 120.21  43.82 133.00  38.12 116.79  33.93 129.53  36.42 147.57  21.33

171.73  23.94 94  0 – 167.07  28.63 161.07  24.99 165.07  15.03 151.50  28.53 157.00  19.21 149.93  14.62

558.80  57.51 328  0 – 533.43  53.34 502.57  41.62 512.36  57.57 486.57  56.71k 494.07  58.33 502.07  46.37

– 58.7 – 95.5 89.9 91.7 87.1 88.4 89.8

171.93  26.01 107  0 9.17  24.52 175.14  16.55 161.29  38.11 164.00  24.11 147.43  43.36 167.93  24.77 166.50  17.39

130.87  18.55 141  0 101.83  46.70 140.71  20.92 138.79  15.20 136.46  13.38 111.71  35.36 130.60  18.57 126.86  18.32

302.80  40.28 248  0 111.00  51.68 315.86  31.75 300.07  44.48 300.46  31.55 268.14  56.25k 298.53  38.28 293.36  31.63

– 81.9 36.7 104.3 99.1 99.2 88.6 98.6 96.9

a

days days days days days days


1–3 0–4 1–5 2–6 3–7 4–8

No statistically significant differences between treatments (one-way ANOVA; P = 0.135). Statistically significant differences between treatments (one-way ANOVA; P = 0.011). Difference between TMP/SQX
1–3 and TMP/SQX 3–7 significant by Tukey’s test (P < 0.05). c Statistically significant differences between treatments (one-way ANOVA; P = 0.021). Difference between UUC and TMP/SQX 0–4 significant by Tukey’s test (P < 0.05); and between UUC and TMP/ SQX 2–6 (P < 0.025). d No statistically significant differences between treatments (one-way ANOVA; P = 0.103). e Statistically significant differences between treatments (one-way ANOVA; P = 0.006). Difference between UUC and TMP/SQX 3–7 significant by Tukey’s test (P < 0.025); and between UUC and TMP/ SQX 2–6 (P < 0.01). f Statistically significant differences between treatments (one-way ANOVA; P < 0.0001). Differences between CUC and all other treatments significant by Tukey’s test (P < 0.001). g Statistically significant differences between treatments (one-way ANOVA; P = 0.003). Difference between CUC and TMP/SQX
1–3 significant by Tukey’s test (P < 0.025). Difference between CUC and TMP/SQX 0–4 significant (P < 0.05). Difference between TMP/SQX
1–3 and TMP/SQX 2–6 significant (P < 0.05). h Statistically significant differences between treatments (one-way ANOVA; P < 0.0001). Differences between CUC and all other treatments significant by Tukey’s test (P < 0.001). Difference between TMP/SQX
1–3 and TMP/SQX 2–6 significant (P = 0.05). i Not included in the statistical analyses because only one bird survived the primary infection on day 0. j CUC = challenged unmedicated control (two groups of same-age birds). k Overall mean weight gains not equal to the totals of mean weight gains during the component time periods because of differences in numbers of birds during some periods. b

R.B. Williams / Veterinary Parasitology 129 (2005) 193–207

Treatment

R.B. Williams / Veterinary Parasitology 129 (2005) 193–207

205

Table 10 Weight gains (S.D.), during 14 days, of chickens infected on day 0 with P. gallinaceum (blood-induced) at 14 days of age in experiment II Treatment

Mean weight gain (g) days 0–7a

Mean weight gain (g) days 7–14b

Mean weight gain (g) days 0–14c

Weight gain days 0–14 as % of UUC

UUC IUC TMP/SQX TMP/SQX TMP/SQX TMP/SQX

88.40  6.22 60.89  17.81 75.33  7.56 77.85  12.71 66.56  17.02 65.20  14.25

114.50  9.40 85.71  19.68 105.13  18.00 110.00  25.79 91.78  34.15 95.67  23.46

202.90  11.76 146.00  30.78 180.47  20.34 187.85  26.41 158.33  28.15 162.33  42.10

– 72.0 88.9 92.6 78.0 80.0

days days days days

4–8 5–9 6–10 7–11

a Statistically significant differences between treatments (one-way ANOVA; P < 0.0001). Difference between IUC and TMP/SQX 5–9 significant by Tukey’s test (P < 0.05). Difference between UUC and IUC significant (P < 0.001). Difference between UUC and TMP/SQX 6–10 significant (P < 0.01). Difference between UUC and TMP/SQX 7–11 significant (P < 0.025). b No statistically significant differences between treatments (one-way ANOVA; P = 0.078). c Statistically significant differences between treatments (one-way ANOVA; P < 0.0001). Difference between IUC and TMP/SQX 4–8 significant by Tukey’s test (P < 0.05); and between IUC and TMP/SQX 5–9 (P < 0.01). Difference between UUC and IUC significant (P < 0.001). Difference between UUC and TMP/SQX 6–10 significant (P < 0.005).

compounds for veterinary use in commercial fowl is clearly P. gallinaceum in the chicken host, as demonstrated herein. The five criteria of efficacy used in the present experiments (mortality, occurrence of green diarrhoea, parasitaemia, haematocrit and body weight gain) were selected from those described by Williams (2005) in an extensive study of the pathology of P. gallinaceum, and the results have confirmed their appropriateness. Mortality and body weight gain are clearly of commercial importance. Feed conversion ratio was not recorded in the present tests because it is a criterion of inconsistent value, particularly for birds that are not completely protected against mortality and reduction of weight gain (Williams, 2005). Production of green diarrhoea, parasitaemia and haematocrit values have proved to correlate with each other very well, as might be expected because they are physiologically related factors in secondary anaemia, which is signalled by biliverdin excretion into the intestine (Williams, 1985). Colonic temperatures were not measured because the febrile effect of P. gallinaceum malaria is fleeting and somewhat inconsistent (Williams, 2005); hence, taking temperatures would not have been useful and would have been unnecessarily stressful for the experimental birds. The TMP/SQX mixture was chosen for assessment in the present study because its safety (Piercy et al., 1984) and some aspects of its bioavailability (Williams et al., 1995) had already been examined in domesticated chickens. Information on its efficacy

against P. juxtanucleare (Shanta et al., 1978), as well as other chicken pathogens, such as the bacteria Pasteurella multocida and Escherichia coli, and several species of eimerian coccidia (White and Williams, 1983), was also available. The likelihood of encountering concurrent malarial (P. gallinaceum and P. juxtanucleare) and coccidial (Eimeria spp.) infections in Asia and Africa is high, and a therapeutic treatment with a wide spectrum of activity, such as TMP/SQX, would therefore be potentially extremely valuable. TMP/SQX has been shown in the present study to have good therapeutic activity against P. gallinaceum. Periods of in-feed medication lasting five days, and beginning on the day before infection or up to four days after infection prevented mortality (Table 1). Birds medicated for periods beginning up to five days after infection had weight gains statistically similar to uninfected controls during the acute phase of malaria (Tables 9 and 10). Furthermore, those birds the treatment of which was begun as late as one or two days after infection exhibited onsets of parasitaemia (Table 4) and production of green diarrhoea (Table 2) that were delayed by 10–13 days and reduced in severity, whilst subnormal haematocrit values (Table 7) were not apparent until 14–17 days later than is usual for infected birds. It therefore seems that TMP/SQX had an inhibitory effect on the development of the parasite, which ultimately was largely eliminated from the blood of the host (Tables 4 and 6). It is likely that the latter effect resulted from a

206

R.B. Williams / Veterinary Parasitology 129 (2005) 193–207

combination of the chemotherapy and the host’s acquired immunity (Tables 5 and 9). The chemotherapeutic protection of birds against malarial infections did not adversely affect acquired protective immunity to a severe challenge 28 days after the primary infection, as judged by mortality (Section 3.1), parasitaemia (Table 5) and weight gain (Table 9). It is difficult to compare directly the effects of TMP/SQX against P. gallinaceum in this study and against P. juxtanucleare in the study of Shanta et al. (1978), because the experimental conditions were so different. In the present study, the total concentration of active ingredients was 332.5 ppm in the feed for five days, beginning at various times before and after infection, whereas Shanta et al. (1978) used 134.4 ppm total active ingredients in the drinking water only for four days beginning on the day of infection. The latter dose level, based upon the calculations of White and Williams (1983) and Williams (1996), is equivalent to approximately 269 ppm in the feed. It may be concluded, therefore, that TMP/SQX had significant activity against P. juxtanucleare (Shanta et al., 1978), at least when administered simultaneously with infection.

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