Perspective Chapter: Application of Probiotics to Inactivate Helminth Parasitic Zoonosis

Abstract

Zoonotic infections may be defined as infections of animals that might be obviously transmissible to people. The contamination may transfer from ingestion of infective level of worms with food, infected soil, skin penetration, or direct animal contact. Parasitic helminths are a group of parasites that remains poorly studied in comparison to viruses and bacteria but may pose a considerable future risk to humans. Zoonotic parasites may be separated into four classes—direct-zoonotic, meta-zoonotic, cyclo-zoonotic, and sapro-zoonotic. Helminth parasitic zoonosis is possible to prevent and manage by simple service of hygiene and sanitation or regular deworming with anthelmintic pills. However, because of the lack of effective vaccines and appeared of anthelmintic resistance to medication, suppression of parasitic infestation still lingers a venture, which needs to improve the new possibility techniques. As a result, the hobby of exploiting probiotics as an alternative to pills has accelerated significantly during the last couple of years. Probiotics are exogenous residing microorganisms, which are beneficial to the host’s fitness when administered inside the digestive tract. The most extensively used microorganisms, for this reason, are microorganisms of the genus Lactobacillus and Enterococcus, and a few fungi and yeasts. The current chapter is proposed to summarize some topics related to the use of probiotics toward helminth parasitic zoonosis.

1. Introduction

Zoonotic infections may be defined as infections of animals that might be obviously transmissible to people. As such, they are worldwide and frequently unfold with humans via their partner and home animals [1, 2]. Zoonotic infections are among the most not unusual on earth and are accountable for over 60% of human infectious sicknesses, some of which can be as a result of helminth parasites. Contamination may also result from ingestion of infective levels of worms with food, infected soil; skin penetration, or direct animal contact. Parasites, such as helminths and protists, are considered pathogenic organisms that occur in developed and developing areas, which are responsible for both foods—water-borne diseases. Their international prevalence is tough to estimate, however the world health organization (WHO) has indicated the worldwide disorder burden of 11 waterborne and foodborne parasitic illnesses, is liable for inflicting over 407 million ailments ensuing in an estimated 94 passable deaths and 11 million disability-adjusted life years [3, 4]. Parasitic helminths are followed by the parasites group that still need more studied as viruses and bacteria, which may pose a future risk to humans. Helminths are macro-parasites, commonly tapeworms (cestodes), roundworms (nematodes), or flatworms (trematodes), and are usually recognized for persistent infections of the gastrointestinal tract, although helminths can infect nearly all human tissues [5, 6].

Helminths include one of the most diverse and geographically widespread groups of parasites that infect humans and animals. Approximately 100 species had been mentioned from humans, generally generating asymptomatic infection or mild signs and symptoms. However, approximately 20 species are of public health significance inflicting severe or maybe fatal infections. Some of the most important and well-known human zoonoses are caused by worm or helminth parasites, including species of nematodes (trichinellosis), cestodes (cysticercosis, echinococcosis), and trematodes (schistosomiasis) [7, 8]. Others include intestinal capillariasis, anisakidosis, eosinophilic enteritis, oesophagostomiasis, and gnathostomiasis [9]. The change of surroundings via wars, famine and the ever expanding and increasingly population brings people into close contact with new environments and flora and fauna species which makes the observe and manage of zoonoses is special interest and complexity [10].

Those zoonotic helminths can cause human diseases and be transferred from consuming food. This food may be meat contaminated with the parasite (taeniasis; trichinosis); fish (diphyllobothriasis; Diplogonorus granidis; clonorchiasis; anisakiasis); invertebrates (paragonimiasis; angiostrongyliasis) or ingestion of the infective degree of the germ with contaminated soil (toxocariasis; hydatid) water or salad (fascioliasis; fasciolopsiasis; hydatid; toxocariasis); pores and skin contact with infected soil/water containing energetic infective larvae and subsequent pores and skin penetration (cutaneous larva migrans; cercarial dermatitis); from direct animal touch (hydatid; toxocariasis) or thru insect vectors/intermediate hosts thru ingestion (dipylidiasis; Hymenolepis diminuta or Inermicapsifer contamination) or injection by way of a mosquito (dirofilariasis; Brugia contamination) [1, 11]. Numerous parasites have been observed on ready-to-consume produce, indicating that modern-day sanitation tactics utilized in the manufacturing of, for example, salads, do not always bring about a product this is free of parasites of fecal origin [12, 13]. This displays that parasite transmission stages within the surroundings are typically incredibly proof against the sanitation approaches normally used inside the food chain. Moreover, as those organisms often have low infectious doses, they may constitute the main danger for customers. A worldwide ranking of foodborne parasites of public fitness significance prepare through the food and Agriculture enterprise (FAO)/WHO become launched in 2014 [14].

Zoonotic parasites may be divided into four classes: direct-zoonotic, meta-zoonotic, cyclo-zoonotic, and sapro-zoonotic. Direct zoonotic parasites infect people directly from animals and involve Entamoeba histolytica, Cryptosporidium parvum, Toxoplasma gondii, and Sarcoptes scabiei. Meta-zoonotic parasites, which contain Fasciola spp. and Schistosoma spp. can infect human beings from invertebrate intermediate hosts. Cyclo-zoonotic parasites have vertebrate intermediate hosts and consist of Echinococcus granulosus, Taenia saginata, and Taenia solium. Saprozoonotic parasites can infect people from soil or water and consist of Ancylostoma caninum and Strongyloides stercoralis [15, 16].

Because of the zoonotic nature of so sufficiently of those food or water-borne parasites, one health recognition technique is needed for controlling and preventing most of those infections. The one health method for tackling zoonotic illnesses desires to take into account the mitigation and prevention of ailment risks that originate at the interface among humans, animals (domestic and wild), and their environments. Accordingly, veterinarians, collectively with other meals and environmental experts worried about the production of food and agricultural and consuming water, play an extensive function in safeguarding food safety [17, 18].

2. Use of probiotics toward helminth parasitic zoonosis

Helminth parasitic zoonosis is simply controlled and prevented by using hygiene and sanitation or regular deworming with anthelmintic pills. However, the vaccines not found to control the helminth parasitic infection, also, the occurrence of anthelmintic medication resistance so, the suppression of parasitic infestation are still needed improvement with new strategies. As a result, the used probiotics was considered the new alternative way instead of on pills or beside the medication which had a significant effect occur in the last couple of years.

Probiotics are anaerobic or aerobic microorganisms found naturally in the raw food and able to isolate simply, which are helpful to the host when consumed with a sufficient amount inside the gastrointestinal tract. These “Good Microorganisms” can be obtained from various dairy products and non-dairy products. The most broadly used microorganisms are used for this purpose, the genus of Lactobacillus and Enterococcus, also, a few fungi and yeasts could be used (Figure 1, [19]). The protective effect of probiotics is by the competition between probiotics and pathogens against colonization or antagonistic elimination within the intestine. An extra mechanism for probiotics is the ability to produce antimicrobial substances, such as bacteriocins or oxygen peroxide, some acids as lactic acid, or through immunomodulation [20, 21]. Similarly, probiotics may also inhibit and opposite the working of parasites inside the intestine. Additionally, their productions could have anthelmintic values and may reduce many parasites’ virulence. Also, probiotic strains play a wide range in the host body as decreasing illnesses and stress, enhancing immunity, modulation of gut microbiota, and nutritional assistance. From this point, probiotics may be used as a new and imperative strategy to control helminth parasites [22].

Figure 1.

Important probiotic strains.

A suitable probiotic strain requirement to confer beneficial belongings (stimulate the immune, antimicrobial activity against pathogens, production of metabolism as bacteriocins and other compounds, and many others.), also, confirmed the probiotic strains to be not pathogenic, able to survive at low pH and acid conditions, thereby continuing within the intestine, and able to colonized and adhered inside the intestine epithelium [23, 24]. Approximately 50 traces similar to 26 species satisfy these standards. Probiotic strains are gram-positive bacteria, isolated from the human intestine microflora or numerous dairy products. However, the beneficial effects of probiotics were more usually tested in model animals than thru direct therapeutic indications and depended largely on the dose consumed. The dose of probiotics is suggested at least 5 billion colonies forming per day for a minimum of 5 days to give their effect [23]. This minimal dose takes under consideration the survival capability of the ingested probiotics inside the gastrointestinal tract, where they may be in opposition with the resident bacteria [25]. Three principal advantages are pronounced: Modulation of the intestinal surroundings, through probiotics having the capacity to manipulate the proliferation of surrounding microorganisms, and/or by using opposition for the occupancy of a common biotope (e.g., get entry to nutriments) [23]. As an example, iron is a proscribing nutriment—it is miles important for maximum bacteria and probiotics can compete for its availability. Lactobacillus can bind ferric hydroxide and reduces iron unavailability for pathogenic microorganisms [26] or by secreting siderophores that chelate and shipping iron [25]. Some probiotics are also capable to influence the composition and balance of the gut microflora [27]. For instance, in probiotic therapy, the use of a mixture of probiotics became shown to grow the whole number of intestinal microorganisms and to repair the variety of the bacterial microbiota in patients [28].

In the end, probiotics also can manage their biotic environment via regulation of intestinal motility and mucus secretion [23, 29]. Secretion of the energetic compounds (such as bacteriocins, antibiotics, loose fatty acids, and hydrogen peroxide) that could manage the boom and/or survival of surrounding microorganisms. Bacteriocins are secreted peptides or proteins that commonly kill closely associated bacteria by using penetrating their membranes or by means of interfering with vital enzymes [28]. Lots of them are produced with the aid of Lactobacillus probiotic lines (lactacin B, lactacin F, nisin, and so forth.). Lactobacillus reuteri produces reuterin (three-hydroxypropionaldehyde), an extensive-spectrum antibiotic, lively against bacteria, yeast, fungi, protozoa, and viruses [30]. By means of lowering the nearby intestinal pH with lactic acid, probiotics can also regulate the increase of acid-sensitive organisms [28]. Also, different probiotics were able to stimulate the reaction of the host immune to an expansion of pathogens to create immunity modulation. Within the intestine, probiotics join with the epithelial cells; Peyer’s patches cells, and immune cells. Those interactions result in a boom within the quantity of IgA producing cells followed by a way of development of IgM and secretory IgA which might be particularly crucial in mucosal immunity, contributing to the barrier against pathogenic organisms [31, 32]. In addition, probiotics can also affect dendritic cells, which might be accountable for the collection of antigens from the intestine and their presentation to native T cells, leading to their differentiation to T-helper (Th1, Th2) or T-regulatory lymphocytes. Probiotic molecules implicated in dendritic cellular induction are poorly characterized, one exception being the S layer protein A of Lactobacillus acidophilus NCFM that regulates maturation of dendritic cells and T cell features [33]. Probiotics have additionally been shown to modulate cytokine release (TNF-α IFN-γ, IL-10, IL-12) [34]. Those cytokines play a valuable function in retaining the delicate balance between important and excessive protection mechanisms. For example, polysaccharide A, synthesized by Bacillus fragilis NCTC 9343, protects in opposition to experimental colitis via an adequate induction of IL-10 manufacturing [35].

3. Some examples for the effect of probiotics in helminth parasitic zoonosis

3.1 Effect of probiotic on Cryptosporidium

Cryptosporidium is an abdominal pathogen followed by the Alveolata group. It could cause overwhelming contamination of gastrointestinal in immunosuppressed humans. In the surroundings, the infective form Cryptosporidium is determined as oocyst found in water. After consumption, the oocysts passage through the gut lumen to the small intestine, in which they release the motile sporozoites that adhere and attack the epithelial intestine cells. The sporozoites are disrupting the microvilli and penetrate to the cells of the host to arrange their intracellular position, wherein they continue to be in a further cytoplasmic vacuole. After replication of parasite and elusion, oocysts are produced and excreted within the faces [36, 37]. Intestinal epithelial cells, inflamed by way of Cryptosporidium parvum, show lessened sodium ions and water absorption as well as greater chloride ion secretion, leading to diarrhea. Also, the use of paromomycin and azithromycin or nitazoxanide is the handiest powerful in mixture with immune restoring agents [38]. Beneficial outcomes of probiotics upon cryptosporidiosis have been tested—female mice were fed day-by-day for 4 weeks vintage with L. reuteri traces 4000 and 4020 or L. acidophilus NCFM provided reduced oocyst dropping [39, 40]. Waters et al. [41] recommended that protection becomes due to secretion of as yet unidentified antimicrobial products. Curiously, in vitro research tested the inhibitory results of cellular-unfastened supernatants of L. acidophilus NCFM and L. reuteri stress 23,272 on C. parvum and C. hominis viability and infectivity [42, 43]. In addition, mobile-unfastened supernatants of Bacillus brevis, Enterococcus faecium, and Pseudomonas alcaligenes reduce C. parvum oocyst persistence by inducing oocyst premature excystation [44, 45]. The compounds at the basis of such inhibition are under investigation. Pickerd and Tuthill, [46] resolved diarrhea due to Cryptosporidium oocysts via probiotic lines Lactobacillus rhamnosus GG 10⁹ CFU/day and Lactobacillus casei Shirota 6.56 X 10⁹ CFU/day during 4-week. Within 10 days of beginning, the pain was decreased and the pattern of stool unfastened from Cryptosporidium after 4 weeks after beginning with probiotics.

Also, Sanad et al. [47] studied the therapeutic effects of daily administration of a mixture of L. acidophilus, L. helveticus, and Bifidobacterium bifidum against C. parvum infection in an immunosuppressed mouse model. The parasite was not achieved by eradication. But, the infection of the probiotics used caused a significant reduction in parasite burden, ultrastructural changes with respect to parasite attachment, internalization into epithelial cells, partial compensation of the mucosal damage caused by the parasite, and an increase in serum level IFN. These results reveal the beneficial effects of probiotics on cryptosporidiosis and suggest that they can help to reduce the risk of serious disease in immunosuppressed patients.

Moreover, Del Coco et al. [48] studied the treated C. parvum infection by oral administration of Enterococcus faecalis CECT 7121 as probiotic strain in immunosuppressed mice. Also, studied the effect of C. parvum infection in the intestinal mucosa and counted at each part of the intestine. The results were established that when both E. faecalis and C. parvum were present in the same intestinal location happened interfered with them. Also, proposing that supplementation of E. faecalis can improve the harmful effects on infection of C. parvum. Also, Glass et al. [42] recognized that L. acidophilus (LA) and L. reuteri (LR) cell-free supernatants able to diminish about 21–42% and 30–35%, respectively of the infection of bovine C. parvum and C. hominis in a cell-culture immunofluorescence (CCIF) test. Moreover, reduction of oocyst viability reached 40–80% at 24 h incubation of bovine C. parvum oocysts in the bacterial cell-free supernatants and this reduction was evaluated by flow cytometric analysis and the infectivity of oocyst reached up to 95% by the CCIF analysis. So, the production of antimicrobial compounds secreted from LA and LR had a harmful effect on bovine C. parvum and C. hominis. Likewise, Khalifa [49] evaluated a study consisting of 70 mice as; 60 mice were infected with Cryptosporidium oocysts and immune-suppressed, other 10 mice were not infected and left immune-competent. Formerly, the mice were divided into three groups; group (1) infected mice were treated with L. casei, group (2) infected mice were treated with yogurt, and group (3) infected mice but not treated as control. The counts of oocyst in the mice stools were determined to evaluate the cryptosporidiosis progress and measured by the developmental stages in histopathological sections of ilea. The results found that the parasitic burden in mice was reduced by regular administration of yogurt and L. casei in comparison with the control group. Moreover, the use of yogurt daily was more effective than L. casei where the yogurt was stopped oocyst shedding previous than L. casei and the counts of oocyst were lesser during the experiment duration in comparison with infected mice that treated with L. casei. Previous studies indicated that the used probiotics are promising and hopeful to control and treated the parasite’s development.

In contracts, Guitard et al. [50] studied the feeding rates with L. casei daily with 2 × 10⁷ CFU before 2 days of the infection until the spontaneous clearance of the parasite. Effects on weight gain, parasite burden, mucosal histology, and production of mucosal cytokines (IFNγ, IL10, and TNFα). The authors also indicated that administration of probiotic strains through the infection course was not significantly affected the weight gain, parasite burden, mucosal damage, or mucosal cytokines kinetics. Overall, the studied model data revealed that the use of L. casei as regular administration was unable to eradicate the parasite. Other studies established that treatment the cryptosporidiosis by probiotic strains did not eradicate the parasite, but resulted in a moderate benefit with a decrease in parasite burden and mucosal damage, and these results were obtained after long-term feeding (7–28 days) or prolonged pre-feeding (≥7 days) before infection [51, 52, 53, 54, 55].

3.2 Effect of probiotics on Giardia

Giardia lamblia (also known as Giardia intestinalis or Giardia duodenalis) is an intestinal pathogenic protozoan parasite belonging to the Diplomonad institution that reasons ∼280 million symptomatic human infections in line with 12 months [56]. This monoxenous waterborne parasite has the capability to contaminate an extensive variety of hosts. To initiate the infection for humans need, 10 environmentally resistant cysts to infect. When the cysts passage through the gastrointestinal, they unlock and replicates to form trophozoites. These trophozoites had the ability to reproduce inside the gut lumen and adhere to the epithelium. These proliferate of trophozoites in the gut was associated with the disorder symptoms, such as watery diarrhea, epigastric pain, nausea, vomiting, and weight drop, which appeared during 6–15 days after cyst consumption, but half of the infections stay asymptomatic. The infection was mainly treated by metronidazole and nitroimidazole, but infections can also solve spontaneously. The immune response T cells, neutrophils, macrophages also with IgM, IgG, and IgA antibodies are major players for the decision of giardiasis. L. casei MTCC 1423 stress as well as E. faecium SF68 were additionally effective in eliminating Giardia contamination from mice [53, 57]. Protection becomes related to a diminution of atrophied villi and infiltrating cells inside the small gut of probiotic-handled mice [57] or with an enhancement of the immune response for the reason that production of specific anti-Giardia intestinal IgA and IgG was noticed in dealing with mice. In vivo experimentation on malnourished mice showed that day-by-day pretreatment with L. casei MTCC1423 effectively decreased severity and period of giardiasis, as compared to non-probiotic-fed malnourished mice [58, 59].

Shukla et al. [60] determined the acid-tolerated strains of probiotics L. casei or Lactobacillus yogurt when found in the gastrointestinal tract. The authors have studied the possibility of these isolates to therapeutic treated the infected mice with the Giardia. After 1 day of Giardia infection, it was found that supplementation of probiotics either L. casei or L. yogurt were eliminated the infection severity comparison with Giardia infected mice. All changes in the Pathophysiologically, the morphological and cellular changes of the small intestine were slightest in treated mice with probiotics in compared to harshly inflamed, edematous, vacuolated epithelial cells in infected mice with Giardia. The results concluded that L. yogurt possessed better probiotic properties and has the possibility to diminish the severity of infection in mice with Giardia. Also, Goyal et al. [61] investigated the efficiency of four probiotic strains (L. rhamnosus GG (LGG), L. acidophilus, L. plantarum, and L. casei) against the murine giardiasis modulation. The daily strain was received around 10⁹ CFU for single animal via orogastric gavage. The more effective strain was LGG, which proved more effect in decreasing the duration of G. lamblia cycle, by eliminating the active trophozoite number in the intestine, increasing cyst excretion, and leading to suppression of the disease around 13 days after trial inoculation. Amer et al. [62] evaluated in vitro and in vivo the beneficial effectiveness of bacteriocins that resulted from new Egyptian probiotic Lactobacilli strains [L. acidophilus (P106) and L. plantarum (P164)] against G. lamblia. The results showed that 50 μg of bacteriocin from L. acidophilus eliminated the trophozoites adherence and the counts around 58.3 ± 4.04%. Oral feeding of 50 μg/mouse of bacteriocin from L. acidophilus every 5 days was able to reduce the density of parasites inside the intestines and enhance the strength of the gut disease system of infected mice. The authors established that bacteriocin from strain L. acidophilus (P106) had a promising potential therapeutic outcome and alternative safety way instead of present commercial drugs to treat G. lamblia.

In the same line, the Bifidobacterium efficiency can be evaluated in an experiment against infected mice with G. lamblia infection. The single-dose about 0.1 ml of Bifidobacterium cells for every day significantly eliminated the G. lamblia cysts shedding in feces, and this infection was disappeared totally at the 5 days of probiotic Bifidobacterium inoculation. Also, in the mice group that used metronidazole, the authors found that Giardia cysts were reduced and infection cured on the day 17th of treatment, in comparison with the control group that showed parasite shedding cysts. Moreover, for histopathological results, in vivo by gut cells, the Bifidobacterium has prevented inflection of the Giardia colonization and able to reduce the infection with this parasite [63]. Generally, the usage of probiotic strains, such as Lactobacillus and Saccharomyces, had a positive influence to reduce gastrointestinal symptoms time and repair the damages, especially for giardiasis. Probiotics had the ability to control the composition of commensal microbiota and balance which lead to therapeutic impact. According to pre-clinical and clinical searches, different probiotic strains can increase the antioxidant capacity, destroy oxidative products, regulate the systemic, and activate the responses of mucosal immune as well as reduce gastrointestinal symptoms time, that lead to protect against mucosal damages that induced by parasites. In addition, they can reduce the G. duodenalis proportion load by directly targeting the parasite. They can produce some anti-giardial factors that feature destroy the parasite’s cellular architecture and suppress the parasite’s proliferation and growth [64, 65].

3.3 Effect of probiotics on Eimeria

Eimeria is an apicomplexan parasite responsible for formed coccidiosis found in poultry, cattle, rabbits, dogs, and cats mainly small animals. A primary parasitic ailment in poultry is avian coccidiosis, with a major economic importance worldwide impact [66]. When the chickens ingest oocysts, it is become inflamed and eventually exocyst to shape sporozoites within the lumen of the top gut. Those sporozoites migrate to their preferred sites of development. They then invade villi enterocytes and undergo a first asexual multiplication, the schizogony, leading to the discharge of numerous merozoites that initiate a 2d schizogony via infecting new epithelial cells. Macro- and microgametes are finally produced, starting up the sexual phase that yields environmental resistant oocysts which might be shed within the feces [67]. Two main ways to control this parasite are by drugs, such as amprolium, halofuginone, and monensin lasalocid, or live vaccines. But stay vaccines against coccidiosis are incredibly effective, primarily based on non-attenuated and attenuated lines.

Probiotic supplementation can enhance performance and help alleviate the negative effects of a mixed Eimeria infection. The study by Ritzi et al. [68] evaluated the effects of probiotics on birds and resistance to a mixed Eimeria infection in commercial broilers. Using treatments, including negative control (non-infected, NEG), positive control (Eimeria-infected, PoS), anticoccidial control (0.01% salinomycin, SAL), irregular high-dose water-applied probiotic (WPI), irregular low-dose water-applied probiotic (WPC), and feed-supplemented probiotic (FSP). On day 15 of the experiment, all birds except those in NEG were treated with a mixed dose of Eimeria acervulina, Eimeria maxima, and Eimeria tenella. Samples of birds feces were collected from day 20 to 24 for counts of the oocyst and evaluated the lesion scores were at day 21. The probiotic groups were comparable with the birds for SAL-treated, except during the 6 days immediately following the Eimeria species challenge, where the SAL birds displayed well performance. The lower duodenal and jejunal lesion scores were found for WPC birds, signifying a healthier intestine and improving the resistance to Eimeria species in comparison with the positive control (PoS). Also, fewer oocysts in the feces were recorded for birds in the WPI treatment, although this was not a trend for all of the probiotic treatment groups. The addition of probiotic secretion compounds containing Pediococcus acidilactici in the ration of the birds before experimental infection with E. tenella resulted in mild improvement in the performance parameters, a slight reduction in lesion score, and in the oocysts count when compared with the birds treated with anticoccidial drugs, but that picture was better than the infected non-treated group. The addition of compounds containing natural microflora (especially those producing lactic acid) to the poultry feed or water to overcome coccidial infection especially [69]. Also, chickens fed on Lactobacillus-based ration showed reduced oocysts output compared to controls after challenge with E. acervulina [70].

Another effect of probiotics was observed on malaria as the recent study by Elli et al. [71] investigated the use of probiotic L. casei in treating malaria in mice with chloroquine. Probiotics in combination with chloroquine showed complete suppression in the parasitemia rate. The data were established by histological observation of two major organs, the liver and spleen. Interestingly, further suppression of parasitemia and hemosiderosis was observed when probiotic was given along with chloroquine. Another author Oliveira-Sequeira et al. [72] have shown a reduction in the number of Strongyloides venezuelensis in infected mice about 33% and egg output upon feeding with probiotic Bifidobacterium animalis, and probiotics was improved the immune responses. A new study has linked the microbiome of the human gut with immunity against malaria infections. Gut probiotics represent innovative tools for malaria prevention and lead the way to novel types of vaccination strategies [73, 74].

Conflict of interest

All authors have no conflicts of interest to disclose.