Veterinary Parasitology 170 (2010) 78–87

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Veterinary Parasitology
journal homepage: www.elsevier.com/locate/vetpar

Vertical migration of Haemonchus contortus infective larvae on

Cynodon dactylon and Paspalum notatum pastures in response
to climatic conditions
Bimal S. Amaradasa a, Robert A. Lane b,*, Ananda Manage c
a
Department of Plant Pathology Physiology and Weed Science, Virginia Tech, 119 Price Hall, Blacksburg, VA 24061, USA
b
Department of Agricultural and Industrial Sciences, Sam Houston State University, P.O. Box 2088, Huntsville, TX 77341, USA
c
Department of Mathematics and Statistics, Sam Houston State University, Huntsville, TX 77341, USA

A R T I C L E I N F O A B S T R A C T

Article history: Observations were made on vertical migration patterns of Haemonchus contortus infective
Received 23 November 2009 larvae on Cynodon dactylon (bermudagrass) and Paspalum notatum (bahiagrass) pastures
Received in revised form 6 January 2010 under summer climatic conditions typical of East Texas. Ten thousand H. contortus infective
Accepted 14 January 2010 larvae (L3) were introduced to 100 cm2 subplots of each pasture species within a plot area of
1 m2. Subplots were inoculated with larvae by applying them in an aqueous medium to the
Keywords: soil or mat beneath the vegetation. Herbage from the inoculated areas was harvested on 5
Haemonchus contortus sampling days over a span of 21 days. L3 recoveries were observed and recorded each day on
Vertical migration
four herbage strata viz. 0–5, 5–10, 10–20 and >20 cm from ground level. The log transformed
Cynodon dactylon
larval recovery data were analyzed for effect of day, stratum, and day  stratum interaction
Paspalum notatum
Infective larvae
for each grass species during two separate experimental periods. Precipitation, relative
humidity and temperature during the study were subjected to correlation and multiple
regression analyses with the larval counts. Significant (P  0.0017) differences were found
for the effect of day on larval recoveries. No significant differences were detected for stratum
or day  stratum interaction effects, though stratum provided a strong indication of
influencing larval recovery. A high positive correlation (0.93) between rainfall and total
average daily larval counts was apparent. The multiple regression analysis did not show
significant results for any of the climatic factors tested. This study showed that the H.
contortus infective larvae can survive beyond 21 days in the soil and infest pasture grasses
when the climatic conditions are favorable. Avoiding use of H. contortus contaminated
pasturelands in summer at the onset of rainfall following a dry spell may effectively reduce
nematode loads in susceptible farm animals. Additional studies should focus on factors
affecting long term L3 survivability, migrational pattern on these and other plant species and
the relationship between climatic factors and larval migration patterns throughout the year.
Total larval recovery of H. contortus in this study was greater in bahiagrass than
bermudagrass. While the design of this study did not allow for testing one pasture species
against another, studies with potted plants would allow for some valid comparisons. Soil
characteristics may also play a role in L3 survival and subsequent migration.
ß 2010 Elsevier B.V. All rights reserved.

1. Introduction

* Corresponding author at: Department of Agricultural and Industrial

Sciences, Sam Houston State University, 1831 University Ave., Thomason
Bldg. 222, P.O. Box 2088, Huntsville, TX 77341, USA.
Tel.: +1 936 294 1225; fax: +1 936 294 1232.
E-mail address: blane@shsu.edu (R.A. Lane).
In the southeastern USA goat rearing has become
popular over the past decade (Terrill et al., 2001). Limited
availability of grasslands for larger, less efficient rumi-
nants, such as cattle, and shifting of consumer demand for

0304-4017/$ – see front matter ß 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.vetpar.2010.01.026
 B.S. Amaradasa et al. / Veterinary Parasitology 170 (2010) 78–87
leaner meats with high nutritional value have contributed
to increased goat meat production and consumption
(Mitcham and Mitcham, 2000, pp. 9–11). In addition
changing demographics in large metropolitan areas of the
USA have resulted in ethnic demand for goat meat and milk
products (Terrill et al., 2001). The preference of goats for a
variety of browse plants not generally favored by cattle and
their potentiality in controlling noxious weeds also have
contributed to the popularity of goats (USDA Electronic
Report from APHIS, 2004, p. 1). However, internal parasites
are of major economic significance to goat enterprises
worldwide, particularly where goats are being grazed on
forbs and grasses (Mitcham and Mitcham, 2000, pp. 120–
133). In particular, the gastrointestinal nematode Hae-
monchus contortus has been responsible for high manage-
ment costs and lower profits for goat production in
confinement and semi-confinement systems in the USA
(Mitcham and Mitcham, 2000, pp. 23–26; Terrill et al.,
2007). Though the most common management practice in
controlling H. contortus is the administration of anthel-
mintics, in recent years many studies conducted in the
southern USA have reported development of resistance to
these chemical treatments (Zajac and Gipson, 2000; Terrill
et al., 2001; Mortensen et al., 2003). The resistance to
anthelmintic compounds has increased the importance of
developing pasture management strategies to control
worm infestations. Since the level of disease depends on
the accidental ingestion of the H. contortus third stage,
infective, free living larvae (L3) by grazing ruminants, most
studies emphasize investigating survival and vertical
migration patterns of L3 on pasture lands. Previous studies
have shown climatic conditions such as rainfall (RF),
relative humidity (RH) and temperature greatly influence
the movement of H. contortus L3 on herbage, particularly
vertical migration (Rees, 1950; Misra and Ruprah, 1972;
Krecek et al., 1991). Insufficient effort has been made to
study the migration and survivability of infective larvae in
response to climatic changes in commonly used pasture
grasses of the southeastern USA, under either controlled or
field conditions.
The purpose of this study was to gain a better
understanding of the survivability and behavior of H.
contortus L3 in pasturelands typical of southeastern Texas.
This study was also conducted to determine whether the
L3 stage of H. contortus has a preferred stratum within
pasture swards of bermudagrass and bahiagrass. Second-
ary objectives were to evaluate how such migration may be
affected by environmental conditions and if the two grass
species tested had any influence on larval migration within
grass swards.
2. Materials and methods
This study was conducted at the Sam Houston State
University Gibbs Ranch in Huntsville, Walker County,
Texas. Field sites of bermudagrass (Cynodon dactylon var.
Jiggs and Cheyenne) and bahiagrass (Paspalum notatum
var. Pensacola) in pastures that had not been grazed by
ruminants during the previous 12-month period were
selected for the experiment. The surface soil at each site
was a fine sandy loam of the Falba series.
79
2.1. Culture of H. contortus third stage larvae
Fresh fecal pellets from a naturally infected donor goat
not recently treated with anthelmintics were used to
culture L3 larvae of H. contortus. The goat feces were
placed in a gauze bag suspended in a humid chamber
(quart canning jar) for 7–9 days to initiate hatching of
larval eggs and development to the L3 infective stage.
Thereafter bags of feces were run overnight through the
Baermann apparatus (Agrios, 2005, pp. 831–832). The
bottom 5–8 ml of larval-containing medium in the
Baermann apparatus was transferred to a 50 ml centrifuge
tube. Larvae were then identified to ascertain the donor
goat was predominantly infected with H. contortus.
Though minor proportions of other unidentified nema-
tode species were present in the final extract, only H.
contortus was enumerated. Larvae in quantities of 10,000
per container were stored in 150 cm2 cell culture flasks
having a shallow layer of 40–50 ml of tap water. Under
aerated conditions larvae could be stored for up to 2
months at room temperature (T. Craig, personal commu-
nication, April 14, 2006).
2.2. Larval identification and enumeration
A key adapted from the MAFF (1971) was used to
identify extracted larvae as H. contortus. Ten to fifteen
times diluted samples were used to enumerate larvae
extracted from the Baermann apparatus. The diluted
samples were enumerated by transferring to a petri dish
with 14 cm diameter having a 2 cm2 inscribed grid and
counting larvae within every square along a randomly
selected diameter. Prior to counting, the petri dish was
agitated for uniform distribution of larvae. A dissecting
microscope was used to aid the enumeration process.
2.3. Extracting
2.3.1. L3 larvae from herbage
The Baermann apparatus technique was used again to
collect H. contortus L3 from herbage. Herbage samples
were placed in a wire mesh basket (stainless steel sink
strainer) with a lining of two layers of facial tissues. The
basket with herbage was placed inside the funnel and
submerged with tap water and kept at room temperature
for 24 h. Nematode larvae were collected as described in
Section 2.1 and transferred to a petri dish similar to the
dimensions mentioned above to enumerate the total
number of larvae in the sample. No dilution of larvae
was necessary. Larvae from twelve randomly selected
squares, representing one half of the petri dish, were
counted to arrive at the total number of larvae in the dish.
2.4. Field experiment
Each experimental site with bermudagrass and bahia-
grass was divided into 30 equal plots (six columns  five
rows) having an area of 1 m2 with a buffer zone of 0.5 m in
width surrounding each plot. Experimental sites were in
accordance to a two-factor factorial design. Randomly
selected areas within plots measuring 100 cm2 in the first,
80 B.S. Amaradasa et al. / Veterinary Parasitology 170 (2010) 78–87

third and fifth columns were treated on 20th of June 2006 protected t-test for least significant difference (LSD) at the
with 10,000 L3 larvae. In late afternoon the larva- 5% level of significance. Correlation coefficients for
containing aqueous medium was applied to the soil or individual climatic variables (temperature, relative
grass mat within the marked 100 cm2 subplot using a fine humidity and rainfall) and larval counts were calculated.
glass pipette. Four to six inoculation spots were selected The collective effect of climatic variables on larval counts
randomly within each marked subplot. Due caution was was analyzed by multiple linear regression. The values of
taken to ensure larvae were not transferred directly to temperature and rainfall for regression analyses were
herbage. The adjacent plots of L3 inoculated columns were derived by averaging hourly data for 24 h prior to the
not treated and remained as controls with 100 cm2 marked herbage sample time. The average relative humidity for the
subplots for control sample collection on the same dates as twelve-hour period (night and early morning) prior to
inoculated grass subplots. Wire survey flags were used to sampling time was considered to be the most critical for
mark the inoculated and non-inoculated 100 cm2 subplots. development of dew or condensation on the leaf tissue,
Larval counts from the herbage at four different strata thus was the calculated value used in the regression
were monitored at predetermined days following analyses. Statistical analyses were conducted using SAS
inoculation. The strata investigated included ground version 9.1 (SAS Institute Inc., NC, USA) and Minitab
level to 5, 5–10, 10–20 and >20 cm from ground level. version 14 (Minitab Inc., PA, USA) statistical packages.
Vegetation within the marked areas was harvested on
the appropriate day beginning at 8:00 a.m. Herbage was 3. Results
harvested at 1.5, 3.5, 7.5, 14.5 and 21.5 days after
inoculation (DAI), with day of harvest being randomly Laval recoveries from inoculated plots, from both
assigned to subplots. Accordingly, at each harvest, 24 grasses differed significantly for day (P  0.0017). The
samples (4 herbage strata in 3 inoculated and 3 control interaction of stratum  DAI was not significant for either
plots) were collected per grass species. As two pasture grass species. Bahiagrass showed a significant larval count
species were tested, a total of 48 samples were collected difference (P < 0.0001) for stratum following the first
on each harvest date. The number of larvae on herbage inoculation (BH1). There were no stratum effects for the
was expressed per kg of herbage dry matter following other three experiments involving bahiagrass and bermu-
the procedure of Krecek et al. (1991). Each experiment dagrass. No L3 larvae were recovered from any of the
was limited to 21.5 days since previous studies indicated control plots. The ANOVA results for larval counts are
limited survival of L3 larvae beyond this point when shown in Table 1. Observations of high numbers of L3 on
inoculated into soil (Lyaku et al., 1988; Krecek et al., both grasses for all experiments corresponded with the
1991; T. Craig, personal communication, April 14, 2006). occurrence of a rainy day or prior influence of substantial
On 30th July, 2006 a second inoculation was carried out rain (Figs. 1 and 2). The mean larval counts are listed for the
using the procedures explained previously. Due to different experiments in Tables 2 and 3. The larval recovery
excessive growth and lodging in the original ‘‘Jiggs’’ numbers are reported on a per kilogram of herbage dry
bermudgrass plot, a new site was selected in a field of matter basis for proper comparison purposes. Actual
‘‘Cheyenne’’ bermudagrass hay pasture a few hundred average L3 recovered from wet grass samples ranged from
meters from the original site for the second inoculation. 0 to 170 per sample (data not shown). During the period of
The original bahiagrass site was used for the second experiment one, average daily temperature fluctuated
inoculation. Data collection was identical to the first between 22 and 29 8C. Average relative humidity for the
experiment. Temperature, relative humidity and rainfall
data were obtained for both experimental periods from
Table 1
the SHSU Gibbs Ranch weather station. Analyses of variance for log transformed larval counts from plots of
bermudagrass and bahiagrass inoculated with H. contortus larvae.
2.5. Statistical analysis
Source DF Type I SS Mean F value Pr > F
square
The log transformed numbers of larvae recovered were
BH1 (Bahiagrass inoculation 1)
analyzed via analysis of variance (ANOVA). The natural
Day (DAI) 4 475.20 118.80 26.71 <0.0001
logarithmic transformation of the larval counts was used Stratum 3 121.72 40.57 9.12 0.0001
to transform the data set to resemble a normal distribution Day  Stratum 12 67.80 5.65 1.27 0.2731
and stabilize the variances (Krecek and Maingi, 2004). For
BH2 (Bahiagrass inoculation 2)
some days specific strata yielded zero larval recovery. Day (DAI) 4 164.46 41.11 6.19 0.0006
Since a value does not exist for natural log zero, a constant Stratum 3 39.26 13.08 1.97 0.1340
of 10 was added to each count to enable the analysis. Log Day  Stratum 12 52.45 4.37 0.66 0.7797
transformed larval counts per kilogram of dry matter were BM1 (Bermudagrass inoculation 1)
analyzed for the 5 collection days using a two factor Day (DAI) 4 347.45 86.86 12.08 <0.0001
factorial model that included DAI, stratum, and the Stratum 3 26.74 8.91 1.24 0.3081
interaction of DAI with stratum. Though conducted on Day  Stratum 12 88.66 7.38 1.03 0.4438

nearly identical soils, the analyses for the two pasture BM2 (Bermudagrass inoculation 2)
species were done separately since they were separated by Day (DAI) 4 76.72 19.18 5.23 0.0017
Stratum 3 25.29 8.43 2.30 0.0920
nearly 2 km. If significant statistical differences were
Day  Stratum 12 51.97 4.33 1.18 0.3290
detected, treatment means were separated using Fisher’s
 B.S. Amaradasa et al. / Veterinary Parasitology 170 (2010) 78–87 81

Fig. 1. Number of L3 larvae recovered from different strata of bahiagrass and bermudagrass during the first trial. The daily rainfall experienced during the
trial is depicted by the line graph.

Fig. 2. Number of L3 larvae recovered from different strata of bahiagrass and bermudagrass during the second trial. The daily rainfall experienced during the
trial is depicted by the line graph.

same period was in the range of 36–87%. The average daily
temperature for experiment two ranged from 26 to 30.5 8C
and the relative humidity varied from 43 to 80%.
Fig. 3 shows the log transformed mean larval counts
observed in different strata for the 5 herbage sampling
days. The larval count of BH1, which showed a significant
difference (P < 0.0001) for stratum, was further analyzed
by means separation. The LSD t-test for mean loge larval
counts showed the first stratum (0–5 cm from soil level) to
be significantly different from the uppermost (>20 cm
82 B.S. Amaradasa et al. / Veterinary Parasitology 170 (2010) 78–87

Table 2
H. contortus mean larval counts (per kilogram of dry matter) on varying bahiagrass strata for two experimental periods.

DAIc Mean larval count (larvae kg dm 1

)
a
BH1 BH2b

0–5 cm 5–10 cm 10–20 cm >20 cm 0–5 cm 5–10 cm 10–20 cm >20 cm

1.5 10,359 2559 328 0 575 634 2627 0

3.5 787 0 0 0 294 0 0 0
7.5 5648 3246 1099 0 6897 8286 16,204 6441
14.5 51,871 57,649 87,722 23,207 0 2300 7397 1626
21.5 41,082 28,702 12,428 635 667 902 621 0
a
Bahiagrass experiment one (larval inoculation was done on 06.20.2006).
b
Bahiagrass experiment two (larval inoculation was done on 07.30.2006).
c
Days after inoculation.

Table 3
H. contortus mean larval counts (per kilogram of dry matter) on varying bermudagrass strata for two experimental periods.

DAIc Mean larval count (larvae kg dm 1

)
a
BM1 BM2b

0–5 cm 5–10 cm 10–20 cm >20 cm 0–5 cm 5–10 cm 10–20 cm >20 cm

1.5 7707 3818 0 0 2420 0 0 0

3.5 14,663 5942 4477 638 0 0 432 0
7.5 6338 745 198 0 8399 670 396 861
14.5 8520 6297 14,522 10,661 0 0 0 0
21.5 26,054 42,495 64,579 34,648 1532 1782 0 0
a
Bermudagrass experiment one (larval inoculation was done on 06.20.2006).
b
Bermudagrass experiment two (larval inoculation was done on 07.30.2006).
c
Days after inoculation.

Fig. 3. Loge mean larval counts of four strata (0–5, 5–10, 10–20 and >20 cm) recorded for 5 herbage sampling days for bahiagrass and bermudagrass
experiments.
 B.S. Amaradasa et al. / Veterinary Parasitology 170 (2010) 78–87 83

Table 4 Table 5
Weather data for Gibbs Ranch, Huntsville, Texas during experiment 1. Weather data for Gibbs Ranch, Huntsville, Texas during experiment 2.

Date Mean Mean Precipitation Date Mean Mean Precipitation

RHa (%) temp (8C) (mm) RHa (%) temp (8C) (mm)

06/20/06b 84.17 24.5 4.06 07/30/06b 57.83 28.2 0.00

06/21/06 71.83 27.1 0.51 07/31/06 58.17 27.8 0.00
06/22/06c 64.00 26.1 0.25 08/01/06c 61.25 27.9 0.00
06/23/06 67.67 26.2 0.00 08/02/06 63.42 28.2 0.00
06/24/06d 56.92 26.4 0.00 08/03/06d 56.67 28.1 0.00
06/25/06 64.08 26.7 0.00 08/04/06 55.25 29.0 0.00
06/26/06 48.92 25.1 0.00 08/05/06 46.83 28.8 0.00
06/27/06 38.67 22.6 0.00 08/06/06 47.83 27.9 1.02
06/28/06e 35.75 23.4 0.00 08/07/06e 57.92 25.8 19.30
06/29/06 39.17 24.6 0.00 08/08/06 80.17 27.5 0.00
06/30/06 36.67 25.7 0.00 08/09/06 63.42 28.8 0.00
07/01/06 44.58 24.0 6.60 08/10/06 54.08 28.5 0.00
07/02/06 74.50 25.1 0.51 08/11/06 58.42 29.0 0.00
07/03/06 71.50 24.4 3.56 08/12/06 54.58 29.1 0.00
07/04/06 79.33 24.5 6.35 08/13/06 52.67 29.0 0.00
07/05/06f 86.75 25.2 17.78 08/14/06f 49.67 29.1 0.00
07/06/06 82.58 26.0 0.00 08/15/06 48.25 29.6 0.00
07/07/06 66.08 26.0 0.00 08/16/06 44.08 30.5 0.00
07/08/06 60.58 27.0 0.00 08/17/06 43.08 28.1 36.58
07/09/06 61.67 25.7 4.06 08/18/06 57.83 28.5 8.64
07/10/06 78.75 28.4 2.03 08/19/06 53.17 28.3 0.25
07/11/06 57.92 28.8 0.00 08/20/06 55.33 29.1 0.00
07/12/06g 60.17 29.1 0.00 08/21/06g 53.42 28.9 0.00
a a
Mean relative humidity for the hours of 9:00 p.m. to 9:00 a.m. Mean relative humidity for the hours of 9:00 p.m. to 9:00 a.m.
b b
inoculation. inoculation.
c c
1st sampling. 1st sampling.
d d
2nd sampling. 2nd sampling.
e e
3rd sampling. 3rd sampling.
f f
4th sampling. 4th sampling.
g g
5th sampling. 5th sampling.

from soil level) stratum (LSD analysis data not shown). The
mean separation by LSD for day effect indicated signifi-
cantly higher larval counts for samples taken on rainy days,
or for sample days influenced by prior precipitation and a
corresponding higher relative humidity. For the first
experiment, 14.5 and 21.5 DAI were significantly different
from the other 3 collection days. In experiment two,
significantly higher larval counts were recorded for 7.5
DAI. Precipitation measurements and the corresponding
influence on larval counts are depicted in Figs. 1 and 2. The
weather data for time periods encompassing the first and
second experiments are given in Tables 4 and 5.
During the experimental period of BH1 and BM1
(bermudagrass after first inoculation), 17.78 mm of rain
was experienced on the 4th sampling day. Further, 6 mm of
precipitation occurred between the 4th and 5th sampling
days. This resulted in significantly higher larval counts in
bahiagrass and bemudagrass for those 2 days. Additionally,
in BH1, the 4th day larval counts were also significantly
higher than the 5th day. During the second trial, 19.3 mm
of rainfall occurred on the 3rd sampling day, resulting in a
significantly greater larval recovery for both grass species.
While higher rainfall totals were recorded between the 4th
and 5th sampling dates for the second experimental period
compared to the first (36.5 mm vs. 23.78 mm), larval
counts on the 5th sampling date were significantly higher
than the 4th in only the first experimental period. This may
possibly be explained by variations in RH. During the first
experiment, the average RH between the 4th and 5th
sampling dates was 66.8% while it was only 50.74% for this
interval in experiment two. The low relative humidity
prevalent between the last two sampling dates of
experiment two might have increased mortality rates of
L3 larvae, resulting in lower levels of recovery from
herbage on the last sampling date.
Neither of the two grass species tested in these trials
appears suited to rapid or easy vertical migration by H.
contortus larvae, since so few larvae were recovered on the
grass blades. It seems apparent larvae were more successful
in upward movement on bahiagrass than bermudagrass
(Table 6). In experiment one, 50% more larvae were
recovered from bahiagrass herbage samples than from
bermudagrass. Though recovery rates for both grasses were
low, in experiment two there was a fourfold increase in
larval recovery from bahiagrass compared to bermudagrass.
Very high positive correlations for rainfall and total
average daily larval counts were apparent for BH1, BH2
(bahiagrass after second inoculation) and BM2 (bermuda-
grass after second inoculation). All three correlation
coefficients exceeded 0.93. The BM1 did not show a high
degree of correlation between these factors. The r values
for temperature or relative humidity were not significant
except in BH1, where RH and larval count was r = 0.81, and
BM1, in which temperature and larval count resulted in
r = 0.75. A multiple regression analysis performed for
climatic variables (temperature, relative humidity and
rainfall) and larval counts did not show significant
probability values for any factor.
Larval counts showed that H. contortus larvae could
survive relatively dry periods for up to 21 days after
84 B.S. Amaradasa et al. / Veterinary Parasitology 170 (2010) 78–87
Table 6
Actual H. contortus larvae recovered from bermudagrass and bahiagrass
herbage samples for two experimental periods.
DAIa Total larval counts of three replicatesb
BM1c BH1d BM2e
1.5 26 51 10
3.5 58 2 2
7.5 23 44 54
14.5 198 1156 0 58
21.5 446 230 4 12
Total recovered 751 1483 70
Percent recovered 0.50 0.99 0.05
a
Days after inoculation.
b
Each replicate was inoculated with 10,000 L3 larvae at the beginning
of the experiment.
c
Bermudagrass study after inoculation 1.
d
Bahiagrass study after inoculation 1.
e contortus colonization on different strata of warm season
Bermudagrass study after inoculation 2.
f
Bahiagrass study after inoculation 2.
inoculation under summer weather conditions prevailing
at Huntsville, Texas. The average temperature during the
first trial was 25.7 8C with a total precipitation of 40.8 mm.
Precipitation was not equally distributed among experi-
mental days and the intensity of rainfall was also highly
variable. The average temperature during the second trial
was 28.5 8C. Precipitation totalled 65.7 mm with 35.6 mm
of rain falling on the 17th day following inoculation.
4. Discussion
This study was conducted under field conditions with
no control of weather conditions. Therefore, the 5 sampling
days of each trial had somewhat different weather
conditions from one to the other. Prior studies provide
strong evidence that precipitation plays a significant role
in larval migration from soil to the herbage under both
field and laboratory conditions (Silangwa and Todd, 1964;
Misra and Ruprah, 1972; Okon and Enyenihi, 1977; Fakae
and Chiejina, 1988). Results from this study are in
agreement with the earlier studies. Herbage sampling
days coinciding with rainy conditions or a moist environ-
ment resulted in peak larval recovery from herbage for
both grass species (Figs. 1 and 2). The LSD means
separation further supported these findings. In the first
experiment, the larval counts of sampling days 4 and 5,
with rainy weather and/or moist conditions, were sig-
nificantly different from the other sampling days
(P  0.001). Similarly, on the 3rd sampling day of experi-
ment two a significantly higher herbage larval count was
recorded (P  0.0017). That day was the only rainy day
coinciding with any of the sampling days during the
second trial. A possible explanation for not observing
significantly high larval numbers on the 5th sampling date
might be prevalence of comparatively low relative
humidity between the 3rd and 5th sampling dates.
Previous studies indicate low relative humidity to either
inhibit movement or decrease the survival of L3 (Barger et
al., 1972; Rees, 1950). For both experimental periods,
average daily temperature ranged between 23 and 31 8C,
indicating that temperature was an insignificant factor
contributing to larval movement in this study. Although
relative humidity did fluctuate widely during some days of
the experimental periods and might have influenced the
mortality of L3 as indicated above, it is more likely rainfall
BH2f was the controlling factor responsible for the observed
19
patterns of L3 movement throughout the summer.
2 The primary objective of this experiment was to
208 determine if L3 larvae have a preferred height of
colonization on pasture swards of different grass species.
The effect of stratum on larval counts in herbage samples
299 was statistically significant only in BH1. In that experi-
0.20
ment, the 0–5 cm stratum had significantly more larvae
than the uppermost stratum. However, though not
significant, BH2 (P = 0.13) and BM2 (P = 0.09) gave a strong
indication of the strata effect on larval numbers (Table 1).
Only a few studies have been published regarding L3 H.
pastures species. Most were conducted either under
laboratory conditions or in pots kept in the field under
hand-watered conditions. A few experiments were carried
out in the field employing irrigated plots. In the Haryana
State of northern India, Misra and Ruprah (1972)
monitored vertical migration of H. contortus L3 larvae on
bermudagrass in outdoor pots and observed L3 migrated
more or less to the same height of 12.5–15 cm every season
(autumn, winter, spring and summer), except for rainy
days when some of the larvae migrated as high as 17.5–
22.5 cm. This study did not indicate any distinct seasonal
effect on migration height of larvae. Across all seasons
66.25% of larvae were recovered from the bottom 2.5 cm of
plant material and 20.66% were recovered from the 2.5–
5 cm grass height. Larval recovery rates reported were
35.2, 27.8, 28.6 and 9.3% in autumn, winter, spring and
summer, respectively. These results however greatly differ
from the laboratory study carried out by Silangwa and
Todd (1964) in which only 2–3% of trichostrongylid L3
were found to migrate onto the grass blades when
inoculated into underlying soil. Of these, 59.2, 26.7, 9.9,
3.4, and 0.8% were found on the first, second, third, fourth
and above the fifth inch height levels from the soil,
respectively. This study was conducted entirely under
laboratory conditions with greater control of different
environmental parameters. According to Misra and Ruprah
(1972) it was weather (day to day variations) rather than
the climate (seasonal changes) that influenced the vertical
migration of H. contortus larvae on the grass. These early
studies did not perform rigorous statistical analyses to
determine if the different larval percentages obtained for
tested strata were significantly different from each other.
O’Connor et al. (2007) conducted a study to determine the
effects of amount, timing and distribution of simulated
rainfall on the developmental success of H. contortus.
Sheep feces containing H. contortus eggs (197,000–465,000
eggs per plot) were deposited onto pasture plots under a
rainfall-activated retractable roof which eliminated inci-
dent rainfall. Differing amounts, timing and distribution of
simulated rainfall were investigated on larval develop-
ment seasonally. There was no effect of rainfall amount (6–
32 mm) on larval development in these experiments. A
negligible number of eggs (0–0.08% of eggs deposited)
developed into L3 larvae. They concluded that moisture
 B.S. Amaradasa et al. / Veterinary Parasitology 170 (2010) 78–87
conditions soon after fecal deposition are key determi-
nants of H. contortus developmental success, with sig-
nificant penalties on development when simulated rainfall
was applied 7 days or more post-deposition and when the
duration of simulated rainfall was short. Larval recovery
from our experiments was also low, ranging from 0.05 to
1%. Though the present study began with L3 larvae instead
of eggs, both studies indicate high mortality of eggs and
(or) larvae before migration to grass blades. The different
recovery rates recorded from the present and previous
studies indicate larval migration to be greatly dependent
on micro- and macro-climatic factors prevailing in the
experimental area. It is quite possible that a number of soil
characteristics play a role as well. Factors such as soil
moisture at time of inoculation, soil texture, biology,
composition, and others could have an impact on larval
survival and subsequent migration to vegetation. Addi-
tionally, laboratory-reared H. contortus larvae may lack
survival mechanisms present in field-hatched ones.
In South Africa, Krecek et al. (1991) conducted research
to determine the effect of time of day, season and stratum
of herbage (upper, lower and mat) and soil on availability
of H. contortus and H. placei L3 larvae on Kikuyugrass
(Pennisetum clandestinum) overseeded with Italian rye-
grass (Lolium multiflorum). Irrigated field plots contami-
nated with lamb and calf feces with a known number of
eggs were used for inoculation. The upper herbage was
designated the top 5–7 cm (10–14 cm above soil) while the
bottom 5–7 cm comprised the lower herbage. Significant
(P < 0.05) differences were found for season, stratum, and
season with stratum interaction for both nematode
species. However, in this study larval recovery of both
parasites from the upper and lower herbage was statis-
tically similar. In contrast, the present study showed an
indication of strata effect on larval counts for BH1, BH2 and
BM2. The disagreement in results may be due to
differences in number of strata analyzed and their relative
heights from the ground level.
It appears, though not statistically testable here, that H.
contortus L3 were more successful migrating upward on
bahiagrass than bermudagrass under identical environ-
mental conditions (Table 6). The total L3 recovered from
bahiagrass in trial one was 1483 vs. 751 recorded for
bermudagrass. In the second inoculation, a total of 299
larvae were recorded on bahiagrass compared to 70
recovered from bermudagrass. Bahiagrass has a coarser
leaf blade texture with more pubescence, possibly aiding in
better retention of a water film on the stem and leaf blades,
favoring larval migration. Further, bahiagrass leaves and
stems arise as a dense cluster from a fabric of rhizomes in the
soil, resulting in a dense leaf canopy. This results in the
sward retaining moisture for a longer period each day than
bermudagrass swards of equal height. The trials carried out
by Silangwa and Todd (1964) examined the effect of external
morphology of grasses on the level of infective trichos-
trongylid larval migration. The trichostrongylids primarily
consisted of H. placei while minor populations of Cooperia
onchophora and Trichostrongylus colubriformis also existed.
They inoculated a known number of L3 larvae onto the soil
surface of three grass species grown in paper pots under
laboratory conditions and recorded the total number
85
recovered from herbage. Tall fescue (Festuca arundinacea)
showed significantly higher larval recovery than smooth
brome (Bromus inermis) under identical conditions. Accord-
ing to the researchers, tall fescue can be easily wetted and
retains moisture better than smooth brome because its
leaves have a highly ribbed upper surface.
Fakae and Chiejina (1988) found a sudden rise in
herbage infestation of trichostrongylid larvae (mainly
Cooperia, Haemonchus and Trichostrongylus spp.) to occur
simultaneously in tested paddocks with the onset of the first
substantial seasonal rains. These results concur with our
study, demonstrating that rainfall influences larval migra-
tion onto the sampled grass plants. The larval migration
influenced by prevailing environmental factors can be used
to manage gastrointestinal Haemonchosis in grazing rumi-
nants. The technique known as ‘‘evasive strategy’’ relies on
moving treated animals to safe pastures just before the
population of infective larvae on the original pasture rise to
dangerously high concentrations (Barger, 1997). Barger et
al. (1994) used rotational grazing for control of gastro-
intestinal nematodes (including H. contortus) of goats in a
wet tropical environment. Since the hatching and develop-
ment of nematodes in contaminated plots was rapid and
continuous, with short survival on pasture (3–7 weeks),
rotational grazing provided an effective method of control-
ling larvae. Using ten plots, with each being grazed for 3.5
days and rested for 31.5 days, they were able to maintain a
mean egg count of less than 1000 eggs per gram in
experimental animals without anthelmintic treatment
throughout the entire study period.
Evasive grazing apparently has potential to control
gastrointestinal nematodes in temperate climates as
well. Eysker et al. (2005) conducted an evasive grazing
experiment with sheep in Netherlands to investigate the
feasibility of this strategy on small ruminant farms. They
found that most pastures grazed in May and June
required at least 3 weeks to develop high pasture
infectivity levels for H. contortus while requiring only 2
weeks on pastures grazed in July, August, and September.
Without rest, pasture infectivity levels for H. contortus
subsequently increased, with the highest levels found
between 5 and 9 weeks on pastures grazed May–June and
between 3 and 9 weeks on pastures grazed in July,
August, and September. Afterward, approximately 3
months of deferment was required to decrease the
pasture infectivity to a low level. Since resting a pasture
for 3 months is generally impractical the authors suggest
using evasive grazing with an integrated control scheme.
Though our experiments did not involve the use of live
test animals, the data indicate that larval ingestion may
be minimal during periods of low rainfall and low
humidity, even in contaminated pastures during the
summer months. However, when rains and high humid-
ity do occur under such conditions, larval ingestion is
likely. Using weather forecasts, we believe managers
could make use of forage from highly contaminated
pastures during hot, dry periods, especially in the
afternoons once any dew has evaporated from the grass
blades.
Based on prior research by Lyaku et al. (1988) and
Krecek et al. (1991) and personal communication with Dr.
86 B.S. Amaradasa et al. / Veterinary Parasitology 170 (2010) 78–87
Thomas Craig, each experiment of this study was limited to
21 days. Lyaku et al. (1988) recovered Haemonchus L3 from
soil samples artificially infected and studied under
laboratory conditions for 3 weeks only. Further, the field
study conducted by Krecek et al. (1991) observed that
larval herbage recoveries were very low or negative after
approximately 3 weeks from first migration. Though we
did not expect to observe high counts of L3 for the last
sampling day (21.5 days after inoculation), results from the
first trial (Table 6) indicate larval survival to easily exceed
3 weeks under field conditions. Reasons for this may
include the prevalence of soil moisture due to rainfall
experienced on days 18 and 19 after inoculation and a high
average relative humidity (66.8% average) during the last
week of that experiment. In trial two the lower average
humidity (50.7%) during the final week of the study likely
created a less favorable environment for larval migration.
However, this study provides evidence of larval surviva-
bility and migration ability beyond 21 days after inocula-
tion of soil under the summer conditions prevailing in
Walker County, Texas. Survival of L3 larvae for a prolonged
period of time may be due to their resistance to desiccation
compared to pre-infective L1 and L2. It is believed that the
L3 survival is attributed in some part to their ability to
migrate to more favorable microenvironments and having
a protective sheath (O’Connor et al., 2006). However, L3 is a
non-feeding stage and higher temperatures increase their
mortality through depletion of energy reserves by
increasing metabolic activity.
The field study by Okon and Enyenihi (1977) using feces
containing H. contortus eggs on pasture at Ibadan, Nigeria
showed no L3 larval development in dry months with
rainfall less than 3 mm in the first 7 days following
contamination. But according to them, L3 larvae already
developed previously could survive such dry spells
effectively since they are more resistant to the influence
of external conditions than the eggs and pre-infective
larvae. They found L3 to survive a minimum of 28 days in
May and a maximum of 63 days in September. Results from
the present study, showing considerable larval numbers
even at 21 days following larval inoculation, support the
findings of Okon and Enyenihi (1977).
5. Conclusion
As expected, the primary implication of this study is
that the onset of measurable precipitation or the
prevalence of high humidity increases infective H.
contortus larval populations (L3) on grass swards of
contaminated pasture fields during the hot, summer
period typical of East Texas. Though not well defined
statistically, this study gave a strong indication of strata
effect on larval numbers. Repeating this experiment with
a simulation of moist field conditions may reveal better
strata effect on larva migrational patterns for the tested
pasture species. One important management practice
supported by this study is the avoidance of grazing
susceptible farm animals on H. contortus contaminated
pasturelands in summer at the onset of rainfall following a
dry spell. This will effectively reduce heavy intake of larval
nematodes.
Additional investigation is warranted in determining
diurnal variation that may exist with regard to L3 migration.
If diurnal variations are obvious and proven, altering the
allowable grazing time during the day to coincide with low
presence of larvae could reduce worm intake even during
warm, moist weather conditions. Utilizing nematode egg-
infected goat feces instead of L3 inoculation in future
experiments would better represent actual field conditions,
though accurate enumeration of unhatched eggs would be
difficult, if not impossible. We believe the field survivability
of parasitic nematodes needs to be determined in this region
by conducting an extended experiment for at least 1 year so
that seasonal variations may be detected.
Of the two pasture species tested here, a higher
percentage of larvae was detected on bahiagrass for both
experimental periods (0.99 verses 0.5 and 0.2 verses 0.05
for experiments 1 and 2, respectively) indicating a possible
grass species effect on larval migration. The morphological
features of bahiagrass that aid in moisture retention are
the likely reason for greater larval migration compared to
bermudagrass. Since experimental plots of the two grass
species were not contiguous, a statistical analysis was
deemed invalid. Thus, the effect of different pasture
species on upward larval movement should be further
tested through the use of pot-grown plants under strictly
controlled environmental conditions.
In contrast to findings of some previous work (Lyaku et
al., 1988) this study shows that the H. contortus infective
larvae can survive beyond 21 days in the soil or mat and
infest pastures when the climatic conditions are favorable.
Acknowledgments
We thank Dr. Thomas Craig, Professor in the Depart-
ment of Pathobiology, College of Veterinary Medicine,
Texas A&M University for his valuable advice and
consultation in research techniques and field experimental
design. We also appreciate the assistance of the manager of
Gibbs Ranch, Dennis Stepp, and other staff members for
their support and contributions in conducting this work.
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