Small lungworms (Nematoda: Protostrongylidae) are nematode parasites of a wide range of domestic and wild mammals. Two types of protostrongylids can be distinguished: the meningeal protostrongylids, which parasitize the central nervous system of multiple cervid species (Samuel
These nematodes are not only the primary infectious agents of the classical verminous pneumonia, but they also make damaged lung tissue susceptible to secondary bacterial infections (Kaufmann, 2013). Symptoms of infection in wildlife are not visibly obvious, and differ from one host to another, and the health impacts vary from sporadic observance upon necropsy (Kabakci
Prevalent worldwide, protostrongylids are more widely studied in domestic ruminants (Kuchboev
This study was performed to fill this gap by exploring protostrongylid infections in three endangered antelope species, which previously inhabited the desert zones of Morocco and North Africa (Loggers
Our objective was to identify the protostrongylid parasite species found in these endangered antelope species, and measure the intensity of infection in each host species. We opted for non-invasive methods to complete this survey. First, we used fecal examination for qualitative (larval morphologic characterization) and quantitative (larval counting) analyses. Second, we used molecular tools (sequencing the second internal transcribed spacer (ITS-2) of the nuclear ribosomal DNA (rDNA)) to identify species of isolated nematode larvae using universal primers (Gasser
Souss-Massa national park (SMNP) is located near the city of Agadir (south of Morocco), extending from Oued Souss estuary in the north, to the Massa estuary in the south. Its center is at 9°40’W 30°5’N. Its climate is semi-arid Mediterranean, with an Oceanic influence. The park is home to a large variety of endemic flora (e.g.
To avoid hybridization related problems between addax and oryx, the animals are isolated from each other in two separate reserves within the SMNP. The addax and part of the dorcas gazelle population reside in the first reserve, called ‹‹Rokein›› while the oryx and the rest of the dorcas gazelle population reside in the second reserve named ‹‹Arrouais››. The estimated population sizes are approximately 230 oryx, 440 addax, and 850 dorcas gazelles.
Individual samples of fresh fecal pellets of correspondent herds were randomly collected from the ground during morning hours, according to their availability, from different sites within the park, and regardless of the sex and age of animals. A total of 180 samples, representing all three species of antelope, were collected between January and July of 2015. All samples were labeled, transported immediately to the laboratory, and refrigerated at 4°C until used.
First stage larvae (L1) were extracted from fecal pellets and counted (larvae per gram of feces: LPG) using the modified Baermann technique described by Cassini
Larvae were recognized as protostrongylid larvae by morphological characteristics, particularly of their tail (Boev, 1975). In general, genera are easily identified (and some genera present only one species). Hence, one of each morphologically unique larva was isolated from each host under inverted microscope, and placed in 20 μl of water in a 0.5 mL tube, and preserved at -20°C for molecular analysis. For phylogenic comparisons, L1 larvae from Moroccan domestic sheep were also isolated.
DNA was extracted according to the tissue protocol using a QIAGEN QIAamp DNA Mini Kit (QIAGEN Lake Constance GmbH, Germany) with a slight modification in the tissue digestion step: 180 μl of ATL buffer and 20 μl of Proteinase K were added to the 0.5 ml tube containing the larva, vortexed and centrifuged for a few seconds, then the contents were transferred to a 2 ml tube containing 0.5 mm Zirconia/Silica beads (BioSpec®, BioSpec Products, Inc., USA) and incubated in a thermal shaker (CAT®-H26) for 1 h at 56°C / 900 rpm. Finally, 200 μl of the digestion product was collected for DNA isolation by the QIAGEN protocol.
PCR assays targeted the internal transcribed spacer (ITS-2) regions of the nematode ribosomal DNA. Universal primers described by Gasser
PCR amplification was performed using the AgPath-ID™ Master Mix kit (Applied Biosystems, Foster City, CA, USA) in a total volume of 50 μl per reaction. Each reaction contained 10 μl of DNA extract, 2 μl of each 10 μM primer, 2 μl of 25X RT-PCR Enzyme Mix, 25 μl of 2X RT-PCR Buffer and 9 μl of water. PCR reactions were carried out in a Bio-Rad iCycler® Thermal cycler with initial denaturation at 94°C for 10 min, followed by 40 cycles (45 seconds each) of denaturation at 94°C, annealing at 55°C and extension at 72°C, with a final extension at 72°C for 5 min. PCR amplicons were visualized by electrophoresis in 2 % agarose gel. Positive samples were identified by the presence of a 425 bp band.
PCR products were submitted to a sequencing service provider (BIO BASIC®, Markham ON, Canada) for purification and Sanger dideoxy sequencing using primers NC1 and NC2. Electropherograms were examined by Sequence Scanner Software v1.0 (Applied Biosystems). The GenBank database was searched for sequences that matched the sequencing results using the BLAST algorithm hosted by the national center for biotechnology information (NCBI) network server (Johnson
Prevalence was expressed as percentages, and LPG values as means ± standard deviations. Differences in prevalence were analyzed using the χ2test (95 % confidence interval), and LPG values among the three populations of antelopes were compared with one-way analysis of variance (ANOVA) and the Newman-Keuls multiple comparison test, with P-values of <0.05 indicating significance (GraphPad PRISM® v5.00 software, USA).
Approval of animal care and use committee was not required; this study did not conduct any animal experiments.
Two distinguishable morphological patterns of L1 larvae were isolated from all three antelope species. The two morphological forms of the isolated larvae match with those of
Protostrongylid L.1 larvae patterns recovered from antelopes,
Parasite larvae counts (LPG), and parasite prevalence values are shown in Table 1. The results of ANOVA show statistically significant differences in LPG values for
Prevalence rates and LPG values extracted from feces of each species of Protostrongylids for the three populations of antelopes.
Addax (n = 60) | Gazelle (n = 60) | Oryx (n = 60) | ||||
---|---|---|---|---|---|---|
M. capillaris | N. linearis | M. capillaris | N. linearis | M. capillaris | N. linearis | |
LPG Mean | 92.94 | 6.02 | 133.09 | 1.37 | 1.48 | 32.81 |
LPG Max | 542 | 40 | 951.7 | 20 | 20 | 838.4 |
LPG Min | 0 | 0 | 0 | 0 | 0 | 0 |
Standard error | 13.42 | 1.70 | 23.09 | 0.44 | 0.50 | 13.99 |
Prevalence % | 98.40 | 60.00 | 96.70 | 23,40 | 28.40 | 90,00 |
LPG values for each parasite in all three antelope populations are compared in Figure 2. The highest LPG for
Comparative LPG means in the all three antelopes for
Comparative prevalence rates across the three antelopes for
Eleven sequences were subjected to similarity searches using the BLAST algorithm of NCBI. The sequences isolated from domestic sheep were confirmed to be
The condensed phylogenetic relationships of Protostrongylids based on ITS-2 sequences of the rDNA, using the Neighbor-Joining method with the Tamura 3-parameter method. The percentages of replicate trees in the bootstrap test (1000 replicates) are shown next to the branches. The analysis involved 14 rDNA ITS-2 nucleotide sequences (5 from antelopes, 4 from local sheep and 5 from Genbank). Evolutionary analyses were conducted in MEGA7.
Phylogenetic analysis of the
Phylogenetic analysis of
The phylogenetic relationships showing topology of Protostrongylids using the Neighbor-Joining method with the Tamura 3-parameter method with the bootstrap test (1000 replicates) shown next to the branches. The analysis involved 10 rDNA ITS-2 nucleotide sequences (2 from antelopes and 8 from Genbank). Evolutionary analyses were conducted in MEGA7.
Our morphological and molecular studies revealed that these three antelope species are obviously infected by two species of protostrongylids:
Prevalence and intensity of infection vary among antelope species as shown previously. Compared to other studies conducted under similar conditions, such as Cassini
This variation in excreted larval load (LPG) depends on many factors. The fecal larval output correlates positively with parasite burden, and is linked to intermediate host (terrestrial mollusks) abundance, which is influenced by climatic parameters (moisture and temperature). Many species of terrestrial gastropods which could be implicated in the ecology of protostrongylids have been identified in the geographical area of Souss-Massa estuaries. These include
For wildlife conservation purposes, we have resorted to introduction, reintroduction, and translocation programs, which will impact the ecology and biodiversity of the parasites they carry (Moir
The results of this current study have become part of a dashboard of health indicators that will serve wildlife management programs in the Souss-Massa National Park for routine monitoring, or for further animal translocation operations. These studies should be extended to examinations of adult parasites in eventual cadavers to characterize adult nematodes in each antelope host, and to the infective larvae in the intermediate hosts to identify snail species implicated in the life cycle of each protostrongylid species.