Observations on the biology of Postharmostomum ntowi Hodasi, 1967 (Trematoda: Brachylaimidae) based on intermediate and definitive hosts found in Nigeria

In a survey of the helminth parasites of anurans from Ase, a location in the freshwater creeks of the Niger Delta of Nigeria, Aisien et al. (2017) recorded the metacercariae of a brachylaimid trematode in the rectum of Amnirana galamensis. Since the stage recovered was the metacercaria, it was doubtful if the frog was the definitive host of the trematode. Previous reports of brachylaimids in the West African sub-region have been in avian hosts. Postharmostomum ntowi, a parasite of the gastrointestinal tract of domestic chickens and guinea fowl was described by Hodasi (1967, 1976) in Ghana. Okon and Enyenihi (1980) reported an unnamed Postharmostomum sp. in the caecum of domestic chicken in Oron, Cross River State of Nigeria. Awharitoma et al. (2003) found metacercariae of a bachylaimid in the land snail Limicolaria aurora collected from locations in the Niger Delta of Nigeria. In vivo cultivation of the metacercariae in domestic chicks yielded adults of Brachylaima fuscatum.

In order to determine the snail intermediate host(s) of the meta-cercariae we recovered from A. galamensis, we examined various land snails found in Ase for the presence of larval brachylaimids, with a view to establishing the roles these snails play in the life cycle of the parasite. We also infected 14 days old chicks of Gallus gallus domesticus with metacercariae recovered from Limicolaria aurora and Archachatina papyracea, to determine whether it was a susceptible host to this trematode. In this paper, we report on the larval stages recovered from the snail intermediate hosts and the adult recovered from experimental hosts and natural infections in free range Gallus gallus domesticus from the study areas.

No ethical approval was required nevertheless we followed the guidelines for Ethical Conduct in the Care and Use of Non-human Animals in Research by the American Psychological Association.

As shown in Table 1, a total of 62 snail specimens were examined for infection with larval brachylaimids, 40 from Ase and 22 from Tombia. The snails examined consisted of A. marginata (n=4), A. papyracea (n=4), L. aurora (n=30), Limicolaria sp. (22) and T. oscitans (2). The prevalence and mean intensity of infection are also shown on Table 1. Of the five land snails examined, all except the semi slug T. oscitans harboured larval brachylaimids. Larval stages encountered included branched sporocysts with cercaria (Figs. 1A and 1B) brevicaudate cercariae (Fig. 1C) and metacercariae (Fig. 1D). Sporocysts were recovered in only three specimens (6.38 %) of Limicolaria spp. (one specimen of L. aurora from Ase and two specimens of Limicolaria sp. from Tombia). While all three larval stages occurred in the Limicolaria spp. only metacercariae were recorded in A. marginata and A. papyracea.

Fig. 1.

Mean measurement ±SD of the larval stages is given in micrometer (μm), followed by the range in parenthesis.

Parasites recovery 7, 14, 21 and 28 days post-infection

A pair of chicks infected with metacercariae recovered from L. aurora was sacrificed on the 7^th, 14^th, 21^st and 28^th day post-infection, respectively, and the developing, mature or gravid parasites recovered. The respective numbers of parasites recovered from the infected chicks are shown in Table 3. All parasites whether juveniles, mature or gravid adults were recovered from the caecum. The recovery rate (RR) ranged from 8 to 19 %.

Table 3.

Parasite recovery from infected chicks 7, 14, 21 and 28 days post-infection.

Chicks	Parasite recovery days post-infection								Total	Recovery rate (%)
	7th day		14th		21st		28th
Set 1	1	15	-	-	-	-	-	-	16	16
Set 2	-	-	1	7	-	-	-	-	8	8
Set 3	-	-	-	-	2	15	-	-	17	17
Set 4	-	-	-	-	-	-	7	12	19	19

The pair of chicks infected with metacercariae from A. papyracea was also sacrificed on the 28^th day post-infection. Five gravid parasites were recovered from the first chick and one from the second (RR, 6 %). No parasites were recovered from the control birds. The measurements of the parasites recovered are shown in Table 4.

Table 4.

Morphometric measurement of Postharmostomum ntowifrom experimental hosts and hosts with natural infections.

Parameters	7 days (n=5		14 days (n=7)		21 days (n=7)		28 days (n=8)		Natural infections (n=11)
	Mean±SD	Range	Mean±SD	Range	Mean±SD	Range	Mean±SD	Range	Mean±SD	Range
Body Length (BL)	2458±202	2164–273	3510±389	2930–4029	5865±1010	4562–7192	6639±865	5561–7726	8639.8±927	7060–10089
Body width	729±45	683–799	899±94.2	766–1032	1517±270	1199–1864	1640±229	1432–2098	2183±363	1765–2731
Forebody length (FBL)	1026±119	866–1199	1246±88	1132–-1365	1755±286	1399–2231	1944±123	1764–2098	2546±219.	2098–2930
FBL as %tage of BL	41.6±1.44	40.0–43.9	35.7±2.59	32.2–39.0	30.1±1.79	26.7–32.1	29.6±2.9	25.9–34.5	29.7±3.0	25.7–34.5
Oral sucker length	446.2±44.7	399.6–499.5	528.0±30.0	466.2–566.1	675.5±65.8	599.4–765.9	720.1±90.7	599.4–832.5	1044.4±60.1	965.7–1165.5
Oral sucker width	456.2±49.4	383.0–499.5	551.8±24.4	532.8–599.4	761.1±107.8	632.7–932.4	786.7±115.3	566.1–932.4	1135.2±113.9	990.0–1365.3
Ventral sucker length	273.4±15.3	254.6–294.8	423.2±210.1	335.0–899.1	480.5±68.9	399.6–566.1	553.6±35.3	499.5–599.4	678.1±84.6	566.1–832.5
Ventra sucker width	273.4±26.1	227.8–294.8	358.0±27.6	321.6–388.6	518.5±76.6	399.6–599.4	545.3±81.4	466.2–732.6	687.2±96.8	532.8–832.5
OSW/VSW ratio	1.7±0.2	1.3–1.9	1.6±0.1	1.4–1.7	1.5±0.1	1.3–1.6	1.5±0.2	1.1–1.7	1.7±0.3	1.4–2.6
Pharynx length	203.7±27.5	174.2–227.8	260.3±24.3	214.4–281.4	399.6±19.2	366.3–432.9	407.9±42.7	366.3–466.2	560.0±66.3	466.2–666.0
Pharynx width	243.9±25.8	214.4–281.4	279.5±27.3	254.6–321.6	383.0±31.9	333.0–432.9	403.8±41.5	333.0–466.6	469.2±54.6	366.6–566.1
Distance between suckers	576.1±87.8	466.2–699.3	718.3±74.1	632.7–832.5	980.0±186.3	765.9–1265.4	1111.4±188.3	799.2–1332.0	1416.±186.2	1065.6–1665.0
Oesophagus length	48.2±20.3	26.8–80.4	38.0±15.7	26.8–67.0	85.6±26.2	66.6–133.2	86.6±38.0	33.3–133.2	92.3±25.1	66.6–133.2
Cirrus length	-	-	-	-	507.8±106.6	399.6–599.4	627.±38.9	566.1–666.0	812.5±137.0	566.1–1032.3
Cirrus width	-	-	-	-	591.1±147.4	432.9–732.6	638.3±53.3	532.8–666.0	865.8±194.2	566.1–1298.7
Anterior testis length	-	-	-	-	541.1±116.6	399.6–666.0	521.7±80.7	432.9–632.7	716.0±160.3	499.5–999.0
Anterior testis width	-	-	-	-	582.8±79.3	499.5–666.0	510.6±71.9	399.6–599.4	899.1±201.6	699.3–1398.6
Posterior testis length	-	-	-	-	557.8±187.1	399.6–765.9	471.8±74.2	399.6–599.4	722.6±115.4	532.8–832.5
Posterior testis width	-	-	-	-	757±298.3	499.5–1032.3	782.6±151.5	499.5–899.1	1115.6±235..6	732.6–1565.1
Ovary length	-	-	-	-	416.3±69.3	333.0–499.5	399.6±75.9	333.0–532.8	559.4±96.5	432.9–732.6
Ovary width	-	-	-	-	474.5±50.0	432.9–532.8	394.1±32.7	366.3–432.9	462.9±90.8	299.7–566.1
Egg length	-	-	-	-	-	-	-	-	30.5±2.5	26.8–34.8
Egg width	-	-	-	-	-	-	-	-	17.2±2.3	13.4–20.1

The prominent features in the parasites recovered 7 days post-infection were the oral sucker, the pharynx, intestinal caecae and the acetabulum. The primordia of the genital organs were present but not measureable at this stage (Fig. 2A). The parasites recovered 14 days post-infection were morphologically similar to the preceding stage (7 days), except that they were larger and the primordia of the reproductive organs were clearly visible and measureable (Fig. 2B). At 21 days post-infection, the testes and the ovary were clearly differentiated; the uterus (without eggs) and the vitellaria were also visible (Fig. 2C). The parasites recovered 28 days post-infection were gravid and the vitellaria which were restricted to the lateral fields in the parasites from 21 days post-infection now filled both the extra-caecal and intra-caecal spaces (Fig. 2D).

Fig. 2.

Brachylaimid infections in free range Gallus gallus domesticus from Ase and Tombia

Adult birds purchased from Ase and Tombia were killed and their intestinal tracts examined for brachylaimid infection. The three birds purchased from Ase were all infected (prevalence, 100 %) with 8, 19 and 6 adult (gravid) parasites (Fig. 3), respectively. Of the 19 worms recovered from chicken number 2, 17 were from the caecum while two were from the rectum. Parasites from hen 1 and the cock were all recovered from the rectum. Measurements for the gravid parasites from Ase are also presented in Table 4.

Fig. 3.

Of the two hens purchased from Tombia, only one was infected with two (2) gravid worms.

The long and complex history of the genus Postharmostomum was summarized by Valadão et al. (2018). The account showed that Postharmostomum was first reported from a young chicken from Italy by Wagener (1852), who misidentified it as Distoma dimorphum Diesing, 1850. The error was corrected six years later by Diesing (1858) who described the parasite found by Wagener (1852) as a new species and named it Distoma commutatum. Thereafter, several reports of the occurrence of the parasite were published in Europe and Africa. Witenberg (1923) described a caecal fluke which was assigned to a new genus and species with the name Postharmostomum gallinum from parasites found in chicken from Turkestan in Central Asia. The parasite was differentiated from D. commutatum on account of the shape of the oral sucker, position of the genital pore and most especially, the anterior extent of the vitellaria, which reach the pharynx in P. commutatum but did not exceed the ventral sucker in P. gallinum. Shortly after its description, P. gallinum was synonymised with P. commutatum as it was believed that Wagener (1852) mischaracterized the vitellaria of P. commutatum, especially as no parasites with vitellaria extending anteriorly to caecal bifurcation were found in the caeca of chickens world-wide, even in areas where P. commutatum was originally described (Pereira & Cuocolo, 1939; Deiana & Arru, 1963). The morphological characteristics of the trematode recovered in this study shows it clearly belongs to the genus Postharmostomum (Brachylaimidae).

From the results obtained in this study, it has been established that Amnirana galamensis was an accidental host of the metacercariae found in its gut, since no further development occurred in this host. Similar accidental infections with brachylaimid metacercariae have been reported in Osteolaemus tetrapsis, the African dwarf crocodile (Enabulele et al., 2013) and the fresh water turtle Pelusios castaneus (Adebayo, 2021). The infection in A. galamensis may have arisen from the consumption of an infected snail intermediate host. Of the five land snails investigated, it was only the semi-slug T. oscitans that did not harbour larval brachylaimids. Even this observation needs further investigation since other slugs have been reported to serve as intermediate hosts of brachylaimid parasites in Africa (Canaris, 1963; Sirgel et al., 2012) and elsewhere (Thiengo & Amato, 1995; Valente et al., 2016). Therefore, despite the negative result recorded in this study, the role of slugs in the transmission of brachylaimids cannot be overlooked. It is either that T. oscitans is not susceptible to the brachylaimid under study or that the number of specimens of this slug examined was too few.

Brachylaimids use two land snails as first and second intermediate hosts, respectively (Lewis, 1969; Mas-Coma & Montoliu, 1986; Lewin, 1992; Cribb & Barton, 1991; Gibson & Bray, 1994). The first snail host according to Mas-Coma and Montoliu (1986, 1987, 1995) is very specific, as brachylaimid species use only one or a very small number of closely related mollusc species inhabiting the local area. Since the cercariogenous sporocysts occurred only in Limicolaria spp. (L. aurora and Limicolaria sp.), these snails must be the first intermediate hosts of the brachylaimid occurring in them. Limicolaria spp. are also known to play this role for other brachylaimids in Africa. For example, Canaris (1963) reported that L. martensiana served as first intermediate host for an undetermined Postharmostomum sp. in Uganda while Awharitoma et al. (2003) reported the occurrence of the sporocysts of Brachylaima fuscatum in L. aurora from Nigeria. The recovery of sporocysts from the digestive glands (hepatopancreas) of Limicolaria is in agreement with observations in other studies (Robinson, 1949; Mas-Coma & Montoliu, 1987; Butcher & Grove, 2001; Gracenea & Gonzalez-Moreno, 2002; Awharitoma et al., 2003; Gonzalez-Moreno & Gracenea, 2006; Segade et al., 2011).

The fact that only three specimens of the 62 snails examined harboured sporocysts confirms the earlier observations by Mas-Coma and Montoliu (1986, 1987, 1995) that in brachylaimid species, the prevalence of cercariogenous sporocysts in the first intermediate host is remarkably low. In brachylaimid life cycles, the first intermediate host can also serve as second intermediate host. We believe that L. aurora from Ase and the Limicolaria sp. from Tombia also serve as second intermediate hosts judging by the high prevalence of metacercaria in these snails (see Table 1). Archachatina spp. (A. marginata and A. papyracea) most probably serve only as second intermediate hosts. The similarity in the size range and morphology of the metacercariae from L. aurora and the unidentified Limicolaria sp. is an indication that they belong to the same species.

It needs to be mentioned here that the larval stages of P. commutatum and P. ntowi share some similarities but also have some differences. Both parasites have branched cercariogenous sporocysts harbouring brevicaudate cercariae. In both species the oesophagus is absent, the caeca is saccular and lateral. However, the cercariae of P. ntowi have longer body size (310 – 415) as against 186 – 193 μm for P. commutatum (Alicata 1940); the cercariae of P. ntowi lack the body spines that occur in P. commutatum; the genital primordia which are visible in the cercaria of P. commutatum (Alicata, 1940) are not detectable at this stage of P. ntowi. Again, except for the differences in body size, metacercariae of both parasites are quite similar: have sinuous intestinal caeca which extend to the posterior of the body; both genital structures are present but not well defined. As remarked by Valadão et al. (2018), who conducted a molecular phylogenetic study of P. commutatum (= P. gallinum), DNA sequences from other isolates of Postharmostomum, (including the one studied here), may yet confirm the cosmopolitan distribution of P. commutatum or reveal a hidden diversity related to this genus of avian trematodes.

The progressive development of the metacercariae of the studied brachylaimid from juveniles to mature adults and finally to gravid worms in Gallus gallus domesticus (Figs. 2 A–D) is an indication that the domestic chick is a susceptible experimental host. However, the recovery of gravid adults from free range Gallus gallus domesticus from Ase and Tombia confirms that these birds are the definitive host of this brachylaimid. Moreover, the infection prevalence in both experimental and naturally infected hosts is very similar.

Results obtained in this study have shown that the parasite recovered from Limicolaria spp. and Archachatina spp. is not B. fuscatum previously recorded by Awharitoma et al. (2003). In contrast, the brachylaimid parasites recovered from experimental and naturally infected domestic chicken from Ase and Tombia is Postharmostomum ntowi described by Hodasi (1967) from domestic chicken and the Guinea fowl in Ghana (Hodasi, 1976). Okon and Enyenihi (1980) provided neither measurements nor illustrations for the Postharmostomum sp. they reported from chickens in Oron, Nigeria but judging from the infection site reported (the caeca), it is likely that this parasite was also P. ntowi. The similarities shared by the two parasite populations from Ghana and Nigeria, respectively, include: elongated body lengths that are in the same size range (see Table 5), intestinal caeca that is continuously sinuous down to the posterior extremity as well as the testes and ovary arrangement (the testes are obliquely tandem while the ovary is opposite the anterior testis). Another shared feature is the anterior reach of the uterus which terminates close to the posterior end of the pharynx. As in the type specimens of P. ntowi, the vitelline follicles are both extra-caecal and intra-caecal, extending anteriorly to a point behind the posterior edge of the ventral sucker and posteriorly to the anterior regions of the ovary and the anterior testis (Fig. 2D and Fig. 3). This finding has shown that P. ntowi is not restricted to Ghana but may be widespread in the West African sub-region. Okon and Enyenihi (1980) remarked that humid climate (as in coastal area of Ghana and Oron in Cross River State of Nigeria), may be a factor in the development and transmission of the parasite. This may be so as Ase, where we recorded this parasite is located in a freshwater creek environment where it rains most of the year. It should therefore be expected that this brachylaimid may be a common parasite of domestic chicken in the humid coastal communities of southern Nigeria.

In conclusion, this study has unravelled the life cycle of P. ntowi and has shown that Limicolaria aurora and the unidentified Limicolaria sp. are the first intermediate host of P. ntowi in Ase and Tombia. The two Limicolaria spp. along with A. marginata and A. payracea serve as second intermediate host for this parasite. Thapsia oscitans is apparently the exception as no infection with larval stages of P. ntowi was recorded in this snail. It has also been shown that the domestic chicken (Gallus gallus domesticus) is not only a susceptible experimental host for P. ntowi, but the definitive host for this parasite with the record of natural infections in free-range birds. Full maturity is attained in about 28 days under experimental conditions with recovery rates ranging from 8 to 19 %. The confirmation of P. ntowi infection in Gallus gallus domesticus in Nigeria extends the geographical range of the parasite. Since according to Hodasi (1976) P. ntowi infection is not restricted to domestic chicken in Ghana, it will be necessary to investigate the host range of this parasite in Nigeria.