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Prevalence and molecular characterisations of gastrointestinal pathogens in sheep, goats and fish from Papua New Guinea

Koinari, Melanie (2014) Prevalence and molecular characterisations of gastrointestinal pathogens in sheep, goats and fish from Papua New Guinea. PhD thesis, Murdoch University.

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Gastrointestinal parasites of livestock cause diseases of important socioeconomic concern worldwide. The two main types of intestinal parasites are helminths and protozoa. They can cause high mortality, reduce production and lead to significant overall economic losses. In tropical and subtropical areas, parasitic nematodes are recognised as a limiting factor to the expansion and improvement of smallholder production of small ruminants. In addition to their veterinary importance, some parasites of domesticated livestock can also be transmitted to companion animals and wildlife, and are a threat to public health.

Little is known of the prevalence of internal pathogens in domesticated animals or wildlife in Papua New Guinea (PNG). There have been various reports on gastrointestinal helminth and Eimeria infections in sheep and goats in PNG. However, those surveys have been restricted in their geographical range and have only been performed on animals raised in government or institutional (either university or agriculture research institution) farms. There is insufficient information on the epidemiology of gastrointestinal parasites infecting sheep and goats in smallholder farms. Fish, both cultured and wild caught, are an important food source in PNG, but there have been few previous surveys of parasite infection in fish. Therefore, the aim of this study was to investigate the prevalence of various pathogens including gastrointestinal nematodes, Eimeria, Cryptosporidium, Giardia and Chlamydia in sheep and goats; and Cryptosporidium, Giardia and anisakids in fish in PNG. A total of 504 faecal samples were collected from sheep (n = 276) and goats (n = 228) in smallholder, government and institutional farms, and a total of 614 fish (25 species) (cultured freshwater n = 132, wild freshwater n = 206 and wild marine n = 276) were sampled from February to August 2011. The study sites for sheep and goats covered a wide geographical area, representing the major sheep and goat farming regions of PNG. Samples were screened using microscopy and molecular techniques.

The overall prevalence of gastrointestinal parasites in sheep and goats in 165 small ruminants (110 sheep and 55 goats) using a modified McMaster technique was 77.6 % (128/165), with 72 % (79/110) of sheep and 89 % (49/55) of goats having one or more species of gastrointestinal parasite. The gastrointestinal parasites found and their prevalences in sheep (S) and in goats (G) were: trichostrongylids 67.3 % (S), 85.5 % (G); Eimeria 17.3 % (S), 16.4 % (G); Strongyloides 8.2 % (S), 23.6 % (G); Fasciola 5.5 % (S), 18.2 % (G); Trichuris 1.8 % (S), 3.6 % (G); and Nematodirus 1.8 % (S), 3.6 % (G). Two additional genera were found in goats: Moniezia (9.1 %) and Dictyocaulus (3.6 %). To identify the pathogenic trichostrongylids, further analysis using a speciesspecific qPCR was performed on genomic DNA from faeces of 263 sheep and 228 goats. The prevalence of each nematode in sheep (S) and goats (G) were: Haemonchus contortus 41.1 % (S) and 41.7 % (G), Teladorsagia circumcincta 21.3 % (S) and 24.1 % (G) and Trichostrongylus spp. 14.8 % (S) and 14 % (G). In addition, a total of 94 samples (mostly Eimeria-positives by microscopy), which included 57 faecal samples from sheep and 37 faecal samples from goats, were screened by qPCR. The prevalence of Eimeria was 64.9 % (37/57) for sheep and 91.9 % (34/37) for goats. This is the first study to quantitatively examine the prevalence of gastrointestinal parasites in goats in PNG. The high rates of parasitism observed in the present study are likely to be associated with poor farming management practises including lack of pasture recovery time, lack of parasite control measures and poor quality feed.

Using molecular tools (nested PCRs), genomic DNA samples from sheep (n = 276), goats (n = 228) and fish (n = 614) were screened for Cryptosporidium spp. and positives were genotyped at the 18S rRNA, 60 kDa glycoprotein (gp60) and actin loci. The overall prevalence for Cryptosporidium at the 18S rRNA locus was 2.2 % (6/276) in sheep, 4.4 % (10/228) in goats and 1.14 % (7/614) in fish. In sheep, C. parvum (subtypes IIaA15G2R1 and IIaA19G4R1), C. andersoni and C. scrofarum were identified. In goats, C. hominis (subtype IdA15G1), C. parvum, C. xiaoi and rat genotype II were identified. In fish, C. hominis (subtype IdA15G1), C. parvum (IIaA14G2R1, IIaA15G2R1 and IIaA19G4R1) and a novel genotype were identified in cultured freshwater (n = 2), wild freshwater (n = 1), and wild marine (n = 4) fish hosts. Cryptosporidium was found in four different fish species; Nile tilapia (Oreochromis niloticus), silver barb (Puntius gonionotus), mackerel scad (Decapterus macarellus) and oblong silver biddy (Gerres oblongus). A novel piscine genotype in fish (piscine genotype 8) was identified in two marine oblong silver biddies and it exhibited a 4.3 % genetic distance from piscine genotype 3 (its closest relative) at the 18S locus. The three subtypes of C. parvum identified in sheep and fish (IIaA14G2R1, IIaA15G2R1 and IIaA19G4R1) were all zoonotic. This is the first report of Cryptosporidium spp. in sheep, goats and fish in PNG. Identification of Cryptosporidium in livestock warrants better care of farm animals to avoid contamination and illness in vulnerable populations. The detection of zoonotic Cryptosporidium in livestock suggests these animals may serve as reservoirs for human infection. Zoonotic Cryptosporidium were identified in fish samples from all three groups (cultured and wild freshwater fish and wild marine fish). The identification of zoonotic Cryptosporidium genotypes in fish is important to public health in PNG and should be further investigated. In particular, detection of Cryptosporidium among fingerlings from aquaculture farms warrants further research to gain a better understanding of the epidemiology of Cryptosporidium infection in cultured fish.

Giardia duodenalis was screened in sheep (n = 276), goats (n = 228) and fish (n = 272) using qPCR targeting the glutamate dehydrogenase (gdh) gene. The overall prevalence of G. duodenalis was 9.1 % (25/256) in sheep, 12.3 % (28/228) in goats and 7 % (19/272) in fish. Although the present study found a low prevalence for G. duodenalis in adult small ruminants and fish, these animals can be considered as sources of contamination for susceptible hosts in PNG, because low numbers of Giardia cysts can cause infection. This is the first study to identify Giardia spp. in sheep, goats and fish in PNG. It demonstrates that G. duodenalis is prevalent in small ruminants and fish in PNG. Giardia duodenalis assemblage E (livestock genotype) was more prevalent in sheep and goats than zoonotic assemblages A and B. Further research into the characterisation of G. duodenalis assemblages in livestock, fish and humans in PNG is required to better understand its zoonotic potential.

The prevalence of anisakid nematode larvae in marine fish collected on the coastal shelves off the townships of Madang and Rabaul in PNG was investigated using microscopy and molecular techniques. The third-stage larvae of several genera of anisakid nematodes are important etiological agents for zoonotic human anisakidosis. Nematodes were found in seven fish species including Decapterus macarellus, Gerres oblongus, Pinjalo lewisi, Pinjalo pinjalo, Selar crumenophthalmus, Scomberomorus maculatus and Thunnus albacares. They were identified by both light and scanning electron microscopy as Anisakis Type I larvae. Sequencing and phylogenetic analysis of the internal transcribed spacers (ITS) and the mitochondrial cytochrome C oxidase subunit II (cox2) gene identified all nematodes as Anisakis typica. This study represents the first in-depth characterization of Anisakis larvae from seven new fish hosts in PNG. The overall prevalence of larvae was low (7.6 %) and no recognised zoonotic Anisakis species were identified, suggesting a low threat of anisakidosis in PNG.

Finally, the prevalence of Chlamydia abortus and C. pecorum in sheep (n = 221) and goats (n = 128) were investigated using two species-specific qPCR assays, targeting the outer membrane protein cell surface antigen gene (ompA). The overall prevalence of C. abortus was 12.2 % (27/221) in sheep and 8.6 % (11/128) in goats, while for C. pecorum, it was 7.7 % (17/221) in sheep and 8.6 % (11/128) in goats. This is the first study to identify C. abortus and C. pecorum in sheep and goats in PNG. It also demonstrates that C. abortus and C. pecorum are prevalent in sheep and goats in PNG, highlighting a need for further research to quantify the production impacts of these bacteria. Furthermore, the results of the present study indicate that C. abortus could pose a risk of transmission to humans, especially, in PNG, where small ruminants in villages are cared for by women on a daily basis.

In summary, the present study represents the most detailed investigation of gastrointestinal parasites using molecular tools in sheep, goats and fish in PNG to date. A variety of gastrointestinal pathogens were identified. This provides an important update on internal pathogens of sheep and goats from previous studies (Quartermain, 2004b). Future investigations should include longitudinal studies and larger cohorts to further assess parasite epidemiology in the diverse agro-climatic zones in the country.

Publication Type: Thesis (PhD)
Murdoch Affiliation: School of Veterinary and Life Sciences
Supervisor: Ryan, Una and Lymbery, Alan
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