Prophylactic and therapeutic methods against histomoniasis in poultry
ISSN 2588-1299
VJAS 2020; 3(2): 593-605
Vietnam Journal
of Agricultural
Sciences
Prophylactic and Therapeutic Methods
Against Histomoniasis in Poultry
Nguyen Van Phuong & Bui Khanh Linh
Faculty of Veterinary Medicine, Vietnam National University of Agriculture, Hanoi
131000, Vietnam
Abstract
Histomoniasis, commonly known as blackhead disease, is a parasitic
disease in poultry caused by the protozoan Histomonas meleagridis.
The availability of various compounds for chemotherapy in the 1970s
resulted in the successful control of blackhead disease. Since the ban
of antihistomonal drugs in the European Union, the disease has re-
emerged, resulting in up to 100% mortality in turkey flocks. This has
renewed the interest of scientists with numerous publications
focusing on prophylactic strategies. This review summarizes the
literature on the preventive and curative options for the control and
treatment of histomoniasis. Two main approaches to the prophylaxis
of the disease were found, which included chemotherapies and plant
substrate products. Histostat-50 and paromomycin were the only
available drugs that showed antihistomonal activity despite some
concern about their threat to human health and antibiotic resistance.
None of the plant substrate products provided potential protection to
birds against blackhead disease. The use of attenuated histomonads
could be an alternative for the prevention of the disease, but the
production of this vaccine prototype is still challenging due to
advanced technique requirements.
Keywords
Histomoniasis, Histomonas meleagridis, parasitic in poultry,
prophylactic, therapeutic
Historical treatment options
Histomoniasis, also known as blackhead disease, histomonosis,
or infectious enterohepatitis, is a parasitic disease caused by the
protozoan Histomonas meleagridis, and was first described by
Cushman (1893). The disease can cause inflammatory lesions in the
caeca and liver of gallinaceous birds, and may lead to flock mortality
of 80-100% in turkey flocks (Swayne et al., 2013). Although
apparent clinical signs are not observed in infected chickens, the
disease can also cause significant mortality in this host (Reece et
al., 1986).
Received: May 15, 2020
Accepted: July 20, 2020
Correspondence to
ORCID
Nguyen Van Phuong
6891
Since the 1970s, histomoniasis has been successfully controlled
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Prophylactic and therapeutic methods against histomoniasis in poultry
by using antiflagellate feed additives such as
dimetridazole and nifursol (Hafez et al., 2005).
In subsequent years, very little was published on
the prevention and treatment of the disease
(Figure 1). Between the 1990s and 2003, the
European Union successively banned all
preventive and curative drugs against H.
meleagridis for use in food-producing animals
because of public concerns about toxicity and
carcinogenicity of the antihistomonal drugs
(Byrne, 2001; Hafez et al., 2005). As a result of
the removal of these treatments, there have been
numerous outbreaks of histomoniasis in the
layer, free-range chicken and turkey flocks,
which have caused considerable economic
losses. A case that demonstrated such losses was
reported in Germany where an outbreak occurred
in a free-range layer flock of hens resulting in 6%
increased mortality and 11% decreased egg
production (Esquenet et al., 2003). Between
2004 and 2008, about 65 flocks in Germany were
infected with H. meleagridis. In 10 farms of
commercial turkeys, a total of 144,000 birds were
detected as infected with the mortality rate rising
by 40% within one week (Hafez et al., 2005;
Hauck et al., 2010).
mortality of 70-80% was recorded in an outbreak
in Thailand in 2006. In 2015, an outbreak of
histomoniasis in free-range chickens with a
mortality of 43.7% was reported in Brazil
(Araújo et al., 2015). The standards for drug
usage differ between regions, and in many cases,
drugs are used without sufficient scientific
evidence of their efficacy.
Another factor complicating the treatment of
histomoniasis was the finding that a strain of H.
meleagridis was partially resistant to nitarsone,
the only approved prophylactic antihistomonal in
the U.S. (Abraham et al., 2014). Additionally,
arsenical residue was recently found in the livers
of roxarsone-treated chickens resulting in the
withdrawal of this substance from the U.S.
market (Kawalek et al., 2011).
This situation has led H. meleagridis to
become of renewed scientific interest, with a
number of recent studies (Figure 1) focusing on
different prophylactic strategies including the
use of chemotherapy drugs such as arsenical,
synthesized nitroheterocylic compounds, and
anthelmintics (van der Heijden, 2009). Plant
substances have also become an alternative
treatment of this parasitic disease. Among all
essential oils and extracts, Natustat and
Protophyt are two commercially available plant
products that have been tested as possible
comments. We acknowledge the insightful
Outbreaks occur not only in such developed
countries but have also been noticed in some
developing countries including Thailand,
Vietnam, and Brazil (Somkid & Niwat, 2006;
Nguyen et al., 2015; Araújo et al., 2015). A
Figure 1. Number of scientific articles on H. meleagridis per year during the last four decades (van der Heijden, 2009)
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594
Nguyen Van Phuong & Bui Khanh Linh (2020)
treatments for histomoniasis (Duffy et al., 2004).
Focusing on immunizations against the disease,
researchers have attempted vaccines with
attenuated cultures of H. meleagridis in vitro and
in vivo. The focus of this review is on
prophylactic and therapeutic options for the
control and treatment of histomoniasis in birds
including chemotherapeutic drugs, plant
substrate products, and vaccinations in the U.S.
and the E.U.
subjected to the successfully tested drugs in the
in vitro test. Dimetridazole, metronidazole,
ornidazole, and tinidazole showed their high
antihistomonal activity at 200-400 ppm, while
paromomycin sulfate and carbadox neither
improved weight gain nor reduced liver and
caecal lesion scores (Hu & McDougald, 2004)
These findings were quite persuasive since the
authors used a strong study design where an in
vitro experiment was followed by an in vivo
experiment for confirmation. The sample size
used in this study was sufficiently reliable (n =
360 birds). Moreover, the dose of H.
meleagridis and the intracloacal inoculation has
successfully been applied by previous
researchers on various occasions (McDougald
& Hu, 2001; Grabensteiner et al., 2006; Zahoor
et al., 2011) which made the findings of this
study more reliable.
Current treatment options
Chemotherapies
Arsenical compounds have been known to
be very effective against histomonad infection by
reducing mortality. However, the concern over
arsenic toxicity resulted in the banning of these
chemotherapies in the European Union in the
period from the 1990s to 2003 (Byrne, 2001).
Following the withdrawal of these compounds,
scientists sought to find new drugs with
antihistomonal properties and began the testing
of various recognized antiprotozoal drugs.
In subsequent years, the prophylactic effect
of paromomycin against histomoniasis was
tested in turkeys (Hafez et al., 2010). Similar to
the methods of Hu and McDougald (2004),
turkeys were challenged with H. meleagridis by
intracloacal infection and then treated with
different concentrations of paromomycin via a
feed additive. These researchers found that the
birds treated with paromomycin had significantly
reduced mortality, reduced liver and caecal
lesion scores, and an increased feed conversion
rate compared to the control birds, indicating that
paromomycin had exhibited its antihistomonal
efficacy (Hafez et al., 2010). This is in line with
the results of a study conducted by Bleyen et al.
(2009) where paromomycin, both in the
commercial form of histoBloc and in drinking
water, showed its preventative effect in 10,000H.
meleagridis intracloacally infected turkeys.
However, this drug did not demonstrate a
curative effect due to the aminoglycoside nature
of paromomycin, which did not allow the drug to
reach the liver stage (Bleyen et al., 2009).
Recently, van de Heijden et al. (2011) confirmed
the effect of this antibiotic in the prevention of
blackhead disease. Both in vitro and in vivo
experiments were carried out with 3 different
concentrations of paromomycin ranging from
100 to 400ppm. Although this antibiotic
suppressed the growth of histomonads at all
In an in vitro experiment realized by Hu and
McDougald (2004), nine antiprotozoal drugs
were tested for activity against H. meleagridis,
namely tinidazole, metronidazole, ornidazole,
and diloxanide furoate, which are known as
compounds active against amoebas and
flagellates; albendazole and mebendazole, which
are anthelmintics with antiprotozoal properties;
and
also
some
antibiotics
including
paromomycin sulfate, quinoline, and carbadox.
Dimetridazole, an effective antihistomonal drug,
which was previously banned in 2003 (Byrne,
2001) was used as a control in the study. In the in
vitro experiment, a dose of 500,000 histomonads
mL-1 was cultured in a modified Dwyer medium
with different concentrations of the tested drugs.
The results showed that growth of H. meleagridis
was completely inhibited at 10 μg mL-1 or higher
of dimetridazole, ornidazole, tinidazole, and
metronidazole. Paromomycin sulfate only
suppressed the growth of the parasite at 100 μg
mL-1 or higher, while other drugs did not show
any antihistomonal effects. These results led to
an in vivo test where chickens, intracloacally
inoculated with 200,000 H. meleagridis, were
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Prophylactic and therapeutic methods against histomoniasis in poultry
concentrations under in vitro conditions, only the
highest concentration provided full protection to
challenged poults (Van de Heijden et al., 2011).
The high dose recommended in this study was
not supported by the European Food Safety
Authority due to the concern of bacterial
resistance (EFSA, 2009). Kempf et al. (2013)
proved the antimicrobial resistance of intestinal
bacteria to paromomycin supplementation in
feed additives in turkeys. Escherichia coli,
Enterococcus faecium, and Staphylococcus
aureus became resistant to paromomycin and
other aminoglycosides in turkeys fed with
paromomycin in feed additives. In addition, E.
coli and E. faecium showed a co-selection for
resistance to other antibiotics (Kempf et al.,
2013).
of tiamulin was assessed. Tiamulin was tested at
different concentrations ranging from 20 to 320
mg L-1. In the experiments, different strains of H.
meleagridis including strains 542/04, 551/04,
and 1169/09 were used in different inoculation
doses from 10,000 to 40,000 parasites mL-1. In
this study, tiamulin showed its inhibitory activity
to histomonal growth at all tested concentrations.
Furthermore, this antibiotic, at all concentrations,
induced lethality to exposed histomonads of the
low dose of inoculation while a high
concentration (320 mg L-1) of this antibiotic
could completely destroy parasites at even a high
dose (40,000 parasites mL-1). This study
demonstrated that tiamulin was effective against
H. meleagridis in vitro. This finding, however, is
not convincing since the doses of inoculation
used in this study were relatively lower than
those used in other studies. The highest dose of
40,000 histomonads mL-1 was lower than 60,000
used in the study of van der Heijden (2009), and
much lower than 500,000 in the experiment of Hu
& McDougald (2004). Moreover, in this study, the
authors pointed out that the effectiveness of the
tested drug decreased when the dose of
inoculation increased. Therefore, the effect of
tiamulin would be questionable with a dose of
inoculation as high as used in other studies.
In essence, paromomycin has been identified
as the most effective antihistomonal drug among
various tested antibiotics despite the concern that
a high dose of this drug could cause intestinal
bacterial resistance in supplemented birds.
Tiamulin was another antibiotic of interest
for scientists during the search for new drugs
effective against blackhead disease. The curative
effect of this drug was reported for the first time
by Burch, who treated infected turkeys with
tiamulin via drinking water at 25 mg kg-1 body
weight for five days (van der Heijden et al.,
2011). In this case, mortality was reduced
A year later, van der Heijden et al. (2011)
performed another in vitro experiment to
evaluate the antihistomonal activity of tiamulin.
Two-fold serial dilutions ranging from 2.5 to 400
μg mL-1 of the antibiotic in PBS were added to
cultures of the parasite. The starting
concentration of histomonads used in the cultures
was 550,000 parasites mL-1. The results obtained
from this experiment showed that the highest
concentration of tiamulin (400 μg mL-1) induced
a slightly lower number of histomonads in the
culture compared to the negative control
cultures. This indicated that the antibiotic was
unable to inhibit the growth of histomonads in
the culture environment. The difference between
the results of this study and Hauck’s could be due
to the variation in the doses of parasites used. In
this study, paromomycin successfully showed its
antihistomonal effect with the same doses of
histomonads used in the tiamulin test. Therefore,
the finding of this study was likely reliable.
significantly,
histomoniasis
and
outbreak
subsequently,
the
was controlled.
According to this report, tiamulin had a
therapeutic effect on blackhead disease, which
shows promise that it could potentially be a new
approach to the treatment of the disease. These
findings, however, would be more convincing if
the authors carried out a thorough microscopical
diagnosis of histomoniasis or prepared a parasite
culture to confirm the presence of H. meleagridis
since caecal necrosis could be a result of
coccidiosis (EFSA, 2009). In addition, it was
likely that the authors ignored the co-effect of
amoxicillin that birds had been previously
supplied. Therefore, it would be beneficial to
perform an in vivo experiment to confirm the
antihistomonal effect of this antibiotic.
Hauck et al. (2010) carried out in vitro
experiments in which the antihistomonal activity
Vietnam Journal of Agricultural Sciences
596
Nguyen Van Phuong & Bui Khanh Linh (2020)
In general, the effect of tiamulin in the
prevention and treatment of histomoniasis is still
a controversial issue. Its antihistomonal effect
was reported in an in vitro experiment by Hauck
(2010), while van der Heijden (2011) claimed the
ineffectiveness of the drug in another test. The
main difference between these two studies was
the dose of the parasites, which probably
explained the contradictory results obtained.
this plant substance product with that of
nitarsone. The results obtained from this study
showed that nitarsone substantially improved
feed conversion ratios and reduced caecal and
liver lesion scores in the nitarsone-supplemented
turkeys compared with the infected non-
supplemented groups. The average body weight
at 42 days was greater in the nitarsone
supplemented group than in the infected non-
supplemented group (Duffy et al., 2005). The
findings of these two studies are not strongly
supported by persuasive evidence, as the major
limitation of these experiments was the
unconvincing design. The authors applied a
natural method of histomonad infection that had
not been tested rigorously by previous
researchers. The authors neither described this
method in detail nor cited its referencing source.
According to Lund et al. (1957) fewer than 0.5%
of embryonated Heterakis gallinae eggs were
found to harbor H. meleagridis infection, and
more than 50% of the worms contained no eggs
with Histomonas spp. Given that using litter
Arsenical Compounds
Nitarsone (4-nitrophenylarsonic acid), a
pentavalent arsenical compound, was at one time
the only available drug on the U.S. market for the
purpose
of
preventing
histomoniasis.
Unfortunately, this drug was withdrawn by the
U.S. Food and Drug Administration at the end of
2015 (Administration USFaD, 2015). There have
been various studies assessing the effectiveness
of this chemical and its commercial form,
Histostat-50, on preventing blackhead disease.
In 2004, scientists from the research team led
by Duffy (2004) compared the antihistomonal
activity of dietary Natustat with Histostat, a
commercial form of nitarsone. Six hundred one-
day-old chickens were divided into four groups
of 150 and then subjected to different treatments.
The first group was the control in which birds
were neither infected with histomonads nor
supplemented with Natustat or Histostat. In the
second group, the birds were given histomonads
but not treatment drugs. Nitarsone at 0.1875
kg/Tone was supplemented to the birds in group
3, whereas Natustat at 1.925 kg/Tone was given
to chickens in group 4. Then the birds in both
groups were experimentally infected with
histomonads. The infection of histomonads was
induced by using litter with a confirmed presence
of Heterakis gallinarum infected by H.
meleagridis. The results of this study illustrated
that the birds treated with nitarsone had
significantly lower cecal and liver lesion scores
compared to those of the infected-non-treated
group, indicating the antihistomonal activity of
this arsenic compound (Duffy et al., 2004). Only
one year later, the authors carried out a similar
study, but this time in male turkeys. Once again,
dietary Natustat for the control of H. meleagridis
was assessed by comparing the effectiveness of
containing
H.
meleagridis-infected
H.
gallinarum to introduce the infection is not a
proven method, the findings of these studies
require further confirmation.
Recently, Abraham et al. (2014) evaluated
the sensitivity of H. meleagridis to nitarsone in
culture media and turkeys. The three strains
collected from different outbreaks were
subjected to nitarsone in culture media. The
results of the in vitro test showed that the strain
of H. meleagridis obtained from the outbreak in
Northern California had sustained resistance to
nitarsone at 100ppm. In the in vivo experiments,
no significant differences between the nitarsone-
treated and the control group were found when
comparing weight gain and caecal and liver
lesions scores. This study illustrates that a strain
of H. meleagridis has obtained partial resistance
to nitarsone. The acceptable sample size and
strong trial control design are supportive of the
results of this study. These findings have raised
concerns from scientists about the resistance of
this parasite to the only permitted
antihistomonal drug.
Following the ban of certain arsenic
compounds, scientists began searching for an
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Prophylactic and therapeutic methods against histomoniasis in poultry
alternative that could protect birds from
histomoniasis. This search focused on the drugs
available for poultry. Various drugs were tested
in vitro, but not many of them successfully
showed antihistomonal activity (Table 2).
by the U.S. Food and Drug Administration at end
of 2015 due to its carcinogenic property.
Plant extracts
As
chemotherapeutics
against
H.
Several
nitroimidazoles
showed
their
meleagridis were banned in the European Union
in 2003, farmers have been left without an
effective treatment against blackhead disease. In
the search for alternative treatments, researchers
have investigated the use of natural plant-based
products.
effectiveness in the protection of birds from
parasites (Table 2). However, they were banned
in the E.U. and the U.S. due to the concern of
carcinogenicity. Histostat-50, the commercial
form of nitarsone, is the only arsenic compound
that has shown potential effectiveness in
preventing the disease. Recently, a strain of H.
meleagridis was found to be partially resistant to
this arsenic compound, which raised the concern
of drug resistance to this unique drug. Among
the various antibiotics tested, only paromomycin
was capable of protecting turkeys from
histomonad infection by reducing the mortality
and the severity of pathological lesions, however,
the use of this drug is tainted by the concern of
antimicrobial resistance. The antihistomonal
effect of tiamulin was still controversial since it
showed a dose-reliant efficacy in in vitro
experiments.
Natustat is
proprietary product containing yeast-derived
mannanoligosaccharide, organic mineral
a
natural, plant-derived
nutrients, and plant extracts, and is used as an
additive to feed for poultry (Duffy et al., 2005).
Various scientists have investigated the
prophylactic effect of this herbal product against
histomonad infections. The ability of Natustat to
manipulate the composition of the intestinal
microflora was proved by using PCR-DGGE
techniques in the study of Waters et al. (2005). In
the caecal contents of turkeys treated with
Natustat, there were various probiotic species
such as Lactobacillus spp. and Bifidobacterium
spp., which may contribute to the success of this
plant-derived product in the control of
histomonad infection.
In brief, Histostat-50 and paromomycin are
the two drugs that have been used in the U.S. for
the prevention of histomoniasis in birds but there
are certain concerns about the use of these drugs
due to drug resistance and human safety.
Histostat was already removed from the market
The antihistomonal effect of Natustat was
assessed in the study of Duffy et al. (2004),
Table 2. Literature evaluating the antihistomonal effects of chemotherapeutic drugs
In vivo (host species)
Product
In vitro
Chicken
Turkey
Diloxanide furoate, albendazole, mebendazole,
quinolone, carbadox
Hu & McDougald
(2004)*
Tinidazole, metronidazole, ornidazole,
dimetridazole
Hu & McDougald
(2004)**
Hu & McDougald
(2004)**
Hafez et al. (2010); Bleyen
et al. (2010);
Hu & McDougald
(2004)
Hu & McDougald
(2004)*
Paromomycin sulfate
van der Heijden (2011)
Duffy et al. (2005);
Histostat-50® (nitarsone)
Duffy et al. (2004)
Abraham et al. (2014)
Note: * Proved ineffective for the protection of birds; ** Banned in the E.U. and the U.S.
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Nguyen Van Phuong & Bui Khanh Linh (2020)
which was evaluated in the section about arsenic
compounds of this review. Among the different
treatment groups, the chicks fed with Natustat
had the heaviest average body weight, highest
food conversion ratio, and lowest mortality.
Further, the caecal lesion scores were
significantly lower than in the nitarsone treated
group. In a similar experiment performed in
turkeys, Natustat fed birds had the same average
body weights as the control (non-infected, non-
supplemented) birds (Duffy et al., 2005).
Although the authors claimed that Natutstat was
effective for the prevention of histomoniasis in
chickens and turkeys, the reliability of these
papers could be questioned due to their
unconvincing study designs.
results of this experiment showed that the three
tested oils had an in vitro antihistomonal activity
despite the absence of synergy among them. A
consistent experimental design, including an
acceptable concentration of incubation (106
parasites mL-1), the determination of the MLC,
and the quantification of live parasites in culture
made the findings of this study reliable.
Three years later, Hafez & Hauck (2006)
continued their interest with these essential oils
by conducting an in vivo study where the
preventive efficacy of Protophyt, a herbal
product containing essential oils of cinnamon,
garlic, lemon and rosemary, against H.
meleagridis infection was assessed. The authors
divided 60 two-week-old turkey poults into three
groups in a controlled trial. Group 1 was
provided Protophyt as a feed additive 6 days
prior to infection until the end of the experiment.
Groups 2 and 3 remained untreated. At 3 weeks
of age, the poults of Groups 1 and 2 were infected
intracloacally with 147,500 H. meleagridis per
bird. Lesion scores of the dead birds and
euthanised birds were recorded. Real-time PCR
was used to confirm the presence of histomonad
DNA in the subjected birds. The results obtained
in the study showed a 20% and 50% mortality
rate in Groups 1 and 2, respectively, while no
deaths occurred in Group 3. In addition, lesions
were found only in caeca and liver, which is
typical for histomonad infection. This study
concluded that Protophyt was an effective
alternative for the reduction of mortality in
turkeys caused by histomoniasis. In this paper,
the authors used a clear study design (control
trial), an acceptable sample size (3 groups of 20
birds each), and a reliable statistic method (t-
test). Also, a molecular technique with ensured
specificity (real-time PCR) was applied to
confirm the presence of the parasite in the
subjected birds, which made these findings
persuasive.
Hauck et al. (2007) investigated the effect of
Natustat in histomonad-infected turkeys. Three
of eight birds in each cage were infected
intracloacally. Heavy paper was used to cover the
floor for one week in order to spread the infection
to the other five birds. In this study, turkey poults
received the Natustat at different rates (250g,
500g, or 1000g per ton of feed). The results
revealed that treatments with Natustat at doses
higher than 250g per ton yielded a significant
reduction in caecal and liver lesion scores, and an
increase in weight gain in indirectly exposed
birds. The authors concluded that Natustat was
effective in controlling the spread of
histomoniasis within the flock. However, it was
noticed that there was a significant difference in
terms of mortality and weight gain between the
directly infected and indirectly infected group,
indicating unsuccessful indirect infections.
These findings did not agree with the previous
study conducted by McDougald & Fuller (2005)
where the authors proved the direct transmission
of histomoniasis among birds.
Zenner et al. (2003) carried out an in vitro
experiment testing the antiparasitic activity of
three essential oil extracts made from fresh
cinnamon leaves, lemon pericarp, and cloves of
van der Heijden & Landman (2008a)
performed a study to examine the in vitro effect
of four herbal products against H. meleagridis,
namely Aromabiotic, Enteroguard, Photophyt B
(drinking water additive), and Photopgyt SP
(feed additive). A H. meleagridis strain was
cultured in modified Dwyer medium prior to the
garlic against H. meleagridis. A dose of 106
-
histomonads mL-1 was used for the anaerobic
incubation, followed by the addition of different
dilutions of the oils to determine the minimal
lethal concentration (MLC). The synergistic
action of the extracts was also analyzed. The
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Prophylactic and therapeutic methods against histomoniasis in poultry
subculture. Dimetridazole and Histostat-50 were
used as positive controls, while PBST was used
as a negative control. Photophyt B at 0.24%
caused a gradual decrease in the number of
parasites over time. By using Enteroguard
suspended at 400 μg mL-1, the decline in the
number of histomonads was observed from 48h.
A significant lower parasite count in comparison
with the negative control was obtained only after
72h of incubation. Aromabiotic and Photopgyt
SP showed no inhibitory effects on histomonad
growth. In this in vitro experiment, the
antihistomonal effects of the herbal products
were not guaranteed since the growth of H.
meleagridis can be inhibited only if the products
were bactericidal to the caecal bacteria present in
the culture of H. meleagridis. Using an antibiotic
induced a complete inhibition of caecal bacterial
growth that resulted in the inhibition of
histomonads in this study. Moreover, the
intestinal flora consisted of various species. A
product may significantly destroy one or more
bacterial species that are essential for histomonad
growth. For these reasons, the results of this
study are not reliable, and might need
confirmation in an in vivo study.
The method of intracloacal inoculation used in
the experiment was likely more reliable in terms
of avoiding re-infection as found in cases of
using litter direct lateral transmission. However,
it is necessary to revise the virulence of the strain
and the dose used in this experiment. The dose of
200,000 histomonads per bird caused mortality
in the group treated with Dimetridazole and
100% mortality in the group of Histostat-50
although these products had been reported as
highly effective drugs against Histomonas spp.
(Hu & McDougald, 2004).
Grabensteiner et al. (2008) performed a
study to investigate the in vitro and in vivo
antihistomonal activities of 43 plant substances
from 18 organic wastes obtained from the food
industry. During the in vitro experiment, only
four ethanolic extracts completely inhibited the
growth of H. meleagridis in the culture with an
initial inoculation of 100,000 histomonads mL-1,
namely the ethanol extracts of saw palmetto,
thyme, pumpkin fruit, and the water and ethanol
extract of grape seed. These four extracts were
selected for the subsequent in vivo experiment.
The concentration of the extracts administered to
the birds was increased from 0.1% to 1% during
the experiment. The birds were experimentally
infected with 103 histomonads/bird via cloaca.
All four tested extracts showed only positive
effects of delaying mortality for up to 18 days in
comparison to the control group. The authors
concluded that none of the tested substances were
able to prevent Histomonas spp. infection in
turkeys. These findings are strongly supported by
an explicit control trial study with a reliable
experimental method and valid in vitro and in
vivo test system. In addition, all data were
analyzed by reliable statistic software.
Following the in vitro experiment, the
authors performed an in vivo experiment with the
same strain of H. meleagridis to confirm the
antihistomonal activity of Enteroguard and
Protophyt. Van der Heijden & Landman (2008b)
conducted the experiment using 234 two-week-
old turkeys divided into 13 groups (n = 18). The
birds were either inoculated with 100, 3162, or
200,000 histomonads per bird. The negative
control group was sham inoculated while the
positive control was provided with no feed
additives, Dimetridazole or Histostat-50. The
study resulted in a mortality rate of 100% in the
birds inoculated with 200,000 histomonads in all
groups except the Dimetridazole-fed group. This
study concluded that Protophyt and Enteroguard
were ineffective at protecting birds from
histomonad infection at a dose of 200,000
parasites/bird. The results obtained are
convincing since the authors had designed a
typical controlled trial with sufficient group sizes
to justify statistical significance. Moreover, the
authors used an adequate range of infection rates.
Responding to the demand of an
antihistomonal alternative, various plant-derived
products have been tested both in vitro and in
vivo. Several plant products successfully
inhibited the growth of H. meleagridis in culture
media such as Enteroguard and several ethanolic
extracts (Table 3). However, none of these
demonstrated dependable protection for birds
from histomoniasis in an in vivo environment. In
three experiments, Natustat showed its
effectiveness against histomoniasis in birds
Vietnam Journal of Agricultural Sciences
600
Nguyen Van Phuong & Bui Khanh Linh (2020)
(Table 3). The fact that these studies used
infective methods made the results controversial.
Therefore, it is recommended that further
research should be conducted to evaluate the
effects of Natustat using different modes of
parasitic infection.
an inactivated vaccine and an attenuated vaccine.
The former was obtained through a freezing and
thawing process and the addition of formol to a
H. meleagridis clonal culture, while the latter
was achieved by passaging the cloned parasites
for around 2 years in intervals of 2-3 days. In this
experiment, 0.5mL of inactivated vaccine was
intramuscularly injected, whereas the live
vaccine was administered cloacally. The
vaccinated birds were subjected to a challenge of
10,000 parasites/bird intracloacally. The turkeys
that received the inactivated vaccine developed
clinical signs and subsequently died, which
indicated that the vaccine did not confer adequate
protection. The birds that received the attenuated
vaccine did not show any clinical signs specific
for histomoniasis. In addition, no parasite DNA
was recovered from the livers of the vaccinated
birds, indicating that the vaccine successfully
protected the birds.
Similarly, controversies about the effects of
Protophyt were found among the studies. This is
one of a very few plant products that has shown
an antihistomonal effect. It would be
worthwhile for researchers to conduct further
studies on this product.
Vaccinations
While chemotherapeutical products for the
control of blackhead disease were used under
high restrictions, and none of the plant-based
substances demonstrated consistent protection
for birds against the disease, vaccination became
a compelling interest for scientists in the quest to
discover an effective treatment and prevention
for histomoniasis.
An immunological study by Bleyen et al.
(2009) was carried out to evaluate the effects of
active and passive immunity in the protection of
In 2008, Hess et al. (2008) investigated the
efficacy of vaccines for the protection of turkeys
from blackhead disease. The authors tested both
turkeys
from
histomoniasis.
Active
immunization was obtained by directly injecting
Table 3. Literature evaluating antihistomonal effect of plant substrate products
In vivo (host species)
Product
In vitro
Chicken
Turkey
van der Heijden &
Landman (2008a)*
van der Heijden & Landman
(2008b)*
Aromabiotic
Enteroguard
van der Heijden et al.
van der Heijden et al. (2008b)*
(2008a)
Duffy et al.
(2004)
Duffy et al. (2005); Waters et al.
(2005); Hauck et al. (2007)
Natustat
Protophyt
B
van der Heijden et al.
van der Heijden et al. (2008b)*
(2008a)
Protophyt
(Essential oils of cinnamon,
garlic, lemon, and rosemary)
van der Heijden et al.
Protophyt
SP
Hafez & Hauck (2006); Van der
(2008a)*
Heijden et al. (2008b)*
Zenner et al. (2003)
Grabensteiner et al.
Ethanol extracts of saw palmetto
Ethanol extracts of thyme
Grabensteiner et al. (2008)*
Grabensteiner et al. (2008)
Grabensteiner et al. (2008)
Grabensteiner et al. (2008)
(2008)
Grabensteiner et al.
(2008)
Grabensteiner et al.
Water and ethanol extracts of grape seed
Ethanol extracts of pumpkin fruit
(2008)
Grabensteiner et al.
(2008)
Note: * Product was confirmed ineffective.
601
Prophylactic and therapeutic methods against histomoniasis in poultry
H. meleagridis antigens intramuscularly or by
experimental infection via the cloaca, while
passive immunization was implemented by
intraperitoneal administration of antisera from
immunized birds. These antisera were previously
assessed for their capacity to destroy H.
meleagridis. In addition, the transfer of antisera
was assessed by measuring titers of anti-H.
meleagridis antibodies. A strong antiparasitic
serum antibody response was observed in both
active immunization groups. On the contrary, the
birds were not protected against a challenge with
3x105 histomonads in the passive immunized
group despite the successful transfer of anti-
parasite antibodies. This indicated that passive
immunization by transferring serum antibodies
did not have a protective effect in turkeys.
vaccine was achieved after 295 passages. The
turkeys were subjected to either virulent
histomonads or attenuated histomonads with a
dose of 104 parasites administered orally,
whereas the chickens were challenged with a
similar infection regime and an additional cloacal
dose. The results showed that liver and caecal
lesions were found in both the chickens and
turkeys receiving virulent infections, while no
changes were observed in the birds infected with
attenuated parasites. Moreover, the virulent
parasites were found in several organs of the
birds, whereas the attenuated isolates were
present only in caecal samples in the PCR test.
This indicated that attenuated H. meleagridis did
not cause any adverse effects to infected chickens
and turkeys. This was one of a very few studies
focusing on vaccinations of this parasite in
chickens. These findings could be useful for
further investigations in the vaccine development
for histomoniasis. However, the design of this
study was not strongly convincing. The authors
used a dose of only 104 histomonads per bird,
which was significantly lower than the dose used
by other scientists, ranging from 1.4x105 to 106
parasites per bird (Zenner et al., 2003; Hu &
McDougald, 2004; Hafez & Hauck, 2006; van
Der Heijden et al., 2008). It could explain why
no changes were observed in the birds infected
by attenuated parasites. Further studies are
required to confirm the effectiveness of the
immunization produced by these attenuated
isolates.
In the study of Liebhart et al. (2010), the
efficacy of oral vaccination of turkeys using
attenuated H. meleagridis was evaluated. The
authors also assessed the negative effects of this
vaccine prototype on the performance of the
birds. Five different groups of birds were used for
the experiments, namely cloacally challenged 2
weeks after being orally vaccinated, cloacally
challenged 4 weeks after being orally vaccinated,
non-vaccinated but inoculum challenged,
vaccinated but not challenged, and non-
vaccinated not-challenged. The results of this
experiment showed that the birds that were
challenged 4 weeks after vaccination, as well as
the vaccinated but not-challenged and the non-
vaccinated not-challenged groups, did not show
any clinical signs related to histomoniasis. All
the birds in the non-vaccinated group and 71.4%
of the birds cloacally challenged 2 weeks after
vaccination contracted blackhead disease.
Moreover, none of the vaccinated but not-
challenged birds showed any abnormal
performance. These findings not only supported
the claim of Hess et al. (2008) about the
effectiveness of the attenuated vaccine against H.
meleagridis but also proved that the vaccine could
be administered orally (Liebhart et al., 2010).
A recent study conducted by Nguyen Pham
et al. (2013) examined the protective
effectiveness of the low-virulent H. meleagridis
against blackhead disease in turkeys. The low-
virulent H. meleagridis was produced by serial
intracloacal passages, which are also known as
back passages. The back passage comprised of an
intracloacal inoculation of histomonad to 3-
week-old birds. Thirteen days post-inoculation,
these birds were euthanized to obtain the parasite
for reinoculation of new birds. An examination
of the virulence of the back passages showed that
the last three intracloacal passages, did not cause
any of the typical lesions found in turkeys.
Subsequently, back passage 10 was used to test
the protective capacity against H. meleagridis.
A study by Liebhart et al. (2010) evaluated
the effects of virulent and attenuated H.
meleagridis on turkeys and chickens. The
virulent histomonads were obtained from clonal
culture after 21 passages, while the attenuated
Vietnam Journal of Agricultural Sciences
602
Nguyen Van Phuong & Bui Khanh Linh (2020)
The birds vaccinated with this low virulent back
passage yielded minor clinical signs, and lower
liver and caecal lesion scores compared to the
unvaccinated challenged group; and no mortality
compared to 71% in the unvaccinated group in an
experimental challenge with a virulent strain of
the parasite. This result indicated that the low-
virulent H. meleagridis attenuated by serial
intracloacally passages was able to protect
turkeys from a virulent strain of this parasite. A
logical experiment design used in this study
made its findings persuasive.
antibiotics showing antiprotozoal activity,
testing plant substances, particularly some
essential oils, and the attempt to develop
effective vaccines.
Among the various drugs tested, Histostat-
50 and Paromomycin are the two available drugs
in some markets that possess antihistomonal
activity despite recently raised concerns about
deleterious side effects such as bacterial
resistance and consumer safety. It is, therefore,
crucial for policymakers to strike a balance
between the benefits of the consumer and the
sustainability of animal production.
In general, most studies evaluating
histomoniasis vaccination were conducted
exclusively in turkeys. The transfer of serum
antibodies from experimentally infected birds
was unable to protect birds from histomonads
infection, while active immunization created by
injecting antigens showed protective activity.
Several authors proved that a vaccination with in
vitro attenuated parasites could provide
protection to turkeys against H. meleagridis
independently of administration (Table 4).
Additionally, this type of vaccination did not
alter the performance of the subjected birds. In a
practical sense, the production of an effective
vaccine would require not only sophisticated
techniques but also a harmonization between
laboratory experiments and field demand.
A large number of plant substances were
tested
for
their
effectiveness
against
histomoniasis, none of which conferred reliable
protection to birds in in vivo experiments. There
was a lack of standardized methodology for
infection among the studies that led to
inconsistent results. It is therefore recommended
to address this issue in further investigations.
Approaches to different types of
immunizations showed that only vaccinations
with attenuated parasites could induce reliable
protection from histomoniasis in turkeys.
However, the production of vaccines,
particularly for this protist, would be challenging
due to a high level of sophisticated techniques
required.
The future of histomoniasis treatments
Acknowledgements
Following the ban of nitroimidazoles,
recognized as high potential antihistomonal
drugs in the European Union and some other
countries, due to the concern of ecological and
human health threats, researchers were forced to
search for viable alternatives in preventing
histomoniasis. In general, three different
approaches were investigated to deal with the
situation. These were the application of
We are grateful for the comments of Dr. Anne
Beasley at the University of Queensland. She also
answered numerous questions about the language
of this paper. We also thank Prof. Nguyen Van Tho
and Dr. Nguyen Thi Hong Chien, our colleagues
at the Vietnam National University of Agriculture,
who also looked over our transcriptions and
provided valuable comments.
Table 4. Literature evaluating the effectiveness of vaccinations in the protection of birds from histomoniasis
Passive vaccination
Active vaccination
Inactivated parasite
Antigens
Attenuated parasite
Hess et al. (2008);
Liebhart et al. (2010);
Bleyen et al. (2009)*
Bleyen et al. (2009)
Hess et al. (2008)*
Nguyen Pham et al. (2013)
Note: * Proved to be ineffective to protect birds from blackhead disease.
603
Prophylactic and therapeutic methods against histomoniasis in poultry
Commission on the preliminary evaluation of the
safety and efficacy of paromomycin sulphate for
turkeys for fattening and turkeys reared for breeding.
The EFSA Journal. 1095: 1-22.
We acknowledge the insightful comments
offered by the anonymous reviewers and the hard
work of the language reviewers who saved us
from many errors.
Esquenet C., De Herdt P., De Bosschere H., Ronsmans S.,
Ducatelle R. & Van Erum J. (2003). An outbreak of
histomoniasis in free-range layer hens. Avian
Pathology. 32(3): 305-308.
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