Histomonosis in poultry: previous and current strategies for prevention and therapy*

Histomonosis is a parasitic disease of poultry with worldwide prevalence. The disease can cause morbidity and mortality in chicken and turkey flocks entailing severe economic losses. In the first half of the last century, there was a high demand to control histomonosis as the turkey industry was severely affected by the disease. Consequently, numerous chemical compounds were tested for their efficacy against Histomonas meleagridis with varying outcomes, that are summarized and specified in this review. At the same time, preliminary attempts to protect birds with cultured histomonads indicated the possibility of vaccination. Several years ago antihistomonal drugs were banned in countries with tight regulations on pharmaceuticals in order to comply with the demand of consumer protection. As a consequence, outbreaks of histomonosis in poultry flocks increased and the disease became endemic again. New approaches to prevent and treat histomonosis are, therefore, needed and recently performed studies focused on various areas to combat the disease, from alternative chemotherapeutic substances to plant-derived compounds until vaccination, altogether reviewed here. Considering existing regulations and the overall outcome of experimental studies, it can be concluded that vaccination is very promising, despite the fact that various challenges need to be addressed until the first ever developed vaccine based upon live flagellates in human or bird medicine can be marketed.     

Differences in the in vitro susceptibility of mono-eukaryotic cultures of <Emphasis Type="Italic">Histomonas meleagridis,Tetratrichomonas gallinarum</Emphasis> and <Emphasis Type="Italic">Blastocystis</Emphasis> sp<Emphasis Type="Italic">.</Emphasis> to natural organic compounds

Currently, all pharmaceuticals for the treatment or prophylaxis of blackhead disease (histomonosis) caused by the flagellate Histomonas meleagridis are banned from the market. Consequently, great interest exists on the finding of alternative drugs for the abatement of histomonosis. In this study, carvacrol, Cassia oil, an essential oil (EO) mixture containing thyme and rosemary EO and a Quillaja saponaria saponin were examined using in vitro assays for antiprotozoal and antibacterial activity testing established against cloned xenic cultures of different isolates of Histomonas meleagridis, Tetratrichomonas gallinarum and Blastocystis sp. Whereas similar minimal lethal concentrations (MLCs) of five Histomonas isolates were obtained for both carvacrol and the EO mixture as well as for the saponin, significantly different MLCs were observed for them with Cassia oil, ranging from 0.25 up to 0.50 μl/ml. Testing the Blastocystis isolates, different MLCs were obtained for all substances, whereas the Tetratrichomonas gallinarum isolates showed identical susceptibilities. The effects are independent of the bacteria, underlining the need of well-defined protozoan cultures for these investigations.     

An in vitro attenuated strain of Histomonas meleagridis provides cross-protective immunity in turkeys against heterologous virulent isolates

In the current study, cross-protective immunity induced by a well-defined clonal strain of Histomonas meleagridis, attenuated by prolonged in vitro cultivation against different clonal heterologous isolates of the same parasite was investigated. For this purpose, 86 turkey poults were assigned to groups consisting of 9–10 birds. Birds of four groups were vaccinated on their 1st day of life followed by re-vaccination on their 14th day of life when the remaining turkeys were left untreated. The challenge was performed using four strains of H. meleagridis that were isolated from chickens or turkeys from different outbreaks of histomonosis in Europe and three of them showed diversities in their genome. Hence, every strain used for the challenge was applied to a group of vaccinated and a group of non-vaccinated birds while birds of the negative control group were sham inoculated. Non-vaccinated birds suffered from severe histomonosis due to the challenge with fatalities reaching from 5 to 10 turkeys per group. Vaccinated birds did not contract clinical signs of the disease following challenge and the increase in weight was unaffected compared to birds of the negative control group. A significant difference in lesion scores was recorded between vaccinated and non-vaccinated groups, with very few instances of liver involvement in the former groups. Livers of vaccinated birds that were without recordable macroscopic lesions were also found negative by immunohistochemical investigation. According to the data obtained, the present study demonstrates, for the first time, the cross-protective capability of a tentative vaccine strain of H. meleagridis attenuated in vitro against heterologous virulent isolates of different origin.     

Oral vaccination of 1-day-old turkeys with in vitro attenuated Histomonas meleagridis protects against histomonosis and has no negative effect on performance

One-day-old turkey poults were vaccinated against histomonosis (syn. histomoniasis) via the oral route by application of in vitro attenuated Histomonas meleagridis. Subsequently, two different groups composed of 14 birds each were challenged cloacally with highly virulent histomonads after 2 or 4 weeks. Two additional groups of non-vaccinated birds were infected with the challenge inoculum at the same time points. In addition, a group of 19 birds, of which 14 were vaccinated but not challenged, were kept for clinical and serological examinations. Non-vaccinated and non-challenged birds (n=10) represented the negative control group. All non-vaccinated but infected birds and 10 out of 14 vaccinated turkeys challenged 2 weeks post vaccination (w.p.v.) contracted severe histomonosis. Turkeys challenged 4 w.p.v. and all remaining birds used in this experiment did not show any pathognomonic clinical signs. In addition, no adverse effect regarding the weight gain could be observed in birds that were vaccinated but not challenged. The excretion of attenuated and virulent live histomonads was observed very infrequently by re-isolation, but transmission to in-contact birds was very efficient. Presence of antibodies was first noticed 3 w.p.v. and antibody levels remained above the cut-off value until termination of the experiment at 16 w.p.v. The present experiment demonstrates for the first time the potential efficacy of in vitro attenuated histomonads used as an orally applied vaccine to 1-day-old turkeys for protection against fatal histomonosis without affecting performance.     

Antiprotozoal activities determined in vitro and in vivo of certain plant extracts against Histomonas meleagridis, Tetratrichomonas gallinarum and Blastocystis sp

A total of 43 plant substances provided as raw material and different kinds of extracts (aqueous, ethanol, and heptane) from 18 different organic wastes obtained from the food/feed industry were investigated for their in vitro activities against clonal cultures of Histomonas meleagridis, Tetratrichomonas gallinarum, and Blastocystis sp. Ethanolic extracts of thyme, saw palmetto, grape seed, and pumpkin fruit proved to be most efficacious. Thus, these extracts were further tested in vivo in turkeys experimentally infected with H. meleagridis by administrating the substances to the birds through the drinking water. Even though a delayed mortality was noticed in some birds medicated with the extracts of thyme, grape seed, and pumpkin fruit, all birds died or had to be euthanized the latest within 5 weeks post infection—with the exception of one bird which was probably never infected with histomonads—due to a severe typhlohepatitis indicative for histomonosis. In addition, none of the substances were able to prevent the spreading of H. meleagridis from infected to in-contact birds. Thus, these studies clearly demonstrate that in vitro studies are of limited value to assess the efficacy of plant substances against histomonosis.     

Loop-mediated isothermal amplification assay for detection of Histomonas meleagridis infection in chickens targeting the 18S rRNA sequences

Histomonas meleagridis is the causative agent of histomonosis, a disease of gallinaceous fowl characterized by necrotic typhlitis, hepatitis, and high mortality. To develop a rapid and sensitive method for specific detection of H. meleagridis, an assay based on loop-mediated isothermal amplification (LAMP) targeting the 18S rRNA gene was established. The detection limit of the LAMP assay was 10 copies for standard plasmids containing an 18S rRNA gene fragment, which was superior to that of a classical PCR method. Specificity tests revealed that there was no cross-reaction with other protozoa such as Trichomonas gallinae, Blastocytis sp, Tetratrichomonas gallinarum, Plasmodium gallinaceum, Toxoplasma gondii, Eimeria tenella, Leucocytozoon caulleryi and Leucocytozoon sabrazesi. The assay was evaluated for its diagnostic utility using liver and caeca samples collected from suspected field cases, the detection rate was 100 and 97.92%, respectively. These results indicate that the LAMP assay may be a useful tool for rapid detection and identification of H. meleagridis in poultry.     

New PA/1220/98-like variant of infectious bronchitis virus in Poland

ABSTRACT

The aim of the present study was to report the first detection of a new infectious bronchitis virus (IBV) variant in Polish commercial flocks which is completely different to any previously known in this region. In 2018, samples from Ross 308 breeding hens aged 35 weeks were delivered for IBV diagnosis. IBV presence was detected, but all attempts to amplify the S gene fragment were negative. The field material was analysed using the Illumina MiSeq platform and a 1073-nt fragment of the S1 coding region was obtained. The gCoV/ck/Poland/516/2018 strain shared only 52.7–58.1% nucleotide identity to any known genotype of IBV and shared the highest identity of 81.4% to the unique North American PA/1220/98 variant. Based on the obtained sequence, a specific molecular test was constructed and used for screening of chicken samples from 35 field cases delivered to our laboratory between 2018 and 2019 for IBV diagnosis. Application of this test enabled detection of another three chicken flocks as positive for this new strain. All positives were identified in commercial layers with egg production problems. To date, the virus has not been detected in broiler chickens. Taking into account the proposed criteria for the definition of a new IBV genotype or lineage, it seems that the detected viruses in Poland, together with the unique North American PA/1220/98 variant, may be classified as separate lineages/genotype in the new IBV classification.

RESEARCH HIGHLIGHTS

• The new IBV variant is distantly related to other known GI–GVII IBV genotypes/lineages.

• It affects long-lived birds causing egg production problems.

• The detected IBV and the unique North American PA/1220/98 variant are candidates for separate lineages in the new GVIII genotype.

     

Pathogenicity and genomic characterization of a novel avian orthoreovius variant isolated from a vaccinated broiler flock in China

Avian orthoreovirus (ARV) infections of broiler flocks cause arthritis/tenosynovitis syndrome and significant economic losses. ARV variants were detected in the USA and Canada. Viral arthritis/tenosynovitis syndrome has occurred frequently in China in recent years. In this study, a variant ARV strain associated with viral arthritis/tenosynovitis syndrome was isolated from broilers and designated as LY383. Genomic sequence and phylogenetic analysis of the σC nucleic acid and amino acid sequences revealed that the isolate was closely related to ARV field strains Reo/PA/Layer/01224B/14, Reo/PA/Broiler/1551/13, GA/14602/2014, GA/13569/2013 and GA/13542/2013, in cluster V, but distinct from most Chinese field strains or commercial vaccine strains. Experimental challenge showed that the isolate could cause arthritis/tenosynovitis syndrome in broilers, which possessed a high level of maternal antibodies induced by commercial ARV vaccines (S1133, 1733 and T98). Furthermore, viral nucleic acid could be detected in cloacal swabs of all challenged birds throughout the entire test from 5?dpi onward. These results suggest that a novel ARV genotype emerges and might become prevalent in broiler flocks in China.

RESEARCH HIGHLIGHTS

• A variant avian orthoreovirus was isolated from a vaccinated broiler flock in North China.

• The ARV field strain was distinct from previous China-origin ARV isolates and vaccine strains.

• The current commercial ARV vaccine could not provide effective protection of broilers against the field isolate infection.

• These findings indicated that variant ARV field strains might become frequent in broiler flocks in China and effective measures should be conducted to prevent and control the disease.

     

Individual and stable autoantibody repertoires in healthy individuals

In the era towards precision medicine, we here present the individual specific autoantibody signatures of 193 healthy individuals. The self-reactive IgG signatures are stable over time in a way that each individual profile is recognized in longitudinal sampling. The IgG autoantibody reactivity towards an antigen array comprising 335 protein fragments, representing 204 human proteins with potential relevance to autoimmune disorders, was measured in longitudinal plasma samples from 193 healthy individuals. This analysis resulted in unique autoantibody barcodes for each individual that were maintained over one year’s time. The reactivity profiles, or signatures, are person specific in regards to the number of reactivities and antigen specificity. Two independent data sets were consistent in that each healthy individual displayed reactivity towards 0–16 antigens, with a median of six. Subsequently, four selected individuals were profiled on in-house produced high-density protein arrays containing 23,000 protein fragments representing 14,000 unique protein coding genes. Based on a unique, broad and deep longitudinal profiling of autoantibody reactivities, our results demonstrate a unique autoreactive profile in each analyzed healthy individual. The need and interest for broad-ranged and high-resolution molecular profiling of healthy individuals is rising. We have here generated and assessed an initial perspective on the global distribution of the self-reactive IgG repertoire in healthy individuals, by investigating 193 well-characterized healthy individuals.

• Highlights

• A unique longitudinal profiling of autoantibody repertoires in healthy individuals

• Autoantibody profiles are highly individual and stable over time

• All individuals display IgG binding to human protein fragments

• The specificity of disease associated autoantigens needs to be thoroughly characterized

• The identification of a small set of highly reactive autoantigens

• Importance of stringent antigen and sample specific cut-offs for defining reactivity

     

Changes in gut bacterial communities in canaries infected by Macrorhabdus ornithogaster

Macrorhabdus ornithogaster is an opportunistic yeast that colonizes the gastric mucosa of many avian species. Until now, no studies have focused on the influence of a gastric infection on the balance of the intestinal microbiota of birds. In this study, 44 faecal samples from individual canaries, with and without M. ornithogaster infection, were analysed. The detection of the yeast was evaluated by 18S rRNA PCR. In order to evaluate the impact of the Macrorhabdus infection on the bacterial communities, culture-independent methods, by the use of amplicon-based sequencing as well as 16S rRNA-DGGE, were adopted. The different health status of animals affected the relative abundance of the main OTUs, with a greater diversification of the gut microbiota in healthy animals compared to the infected. In particular, Lactococcus, Pseudomonas, Acinetobacter, Lachnospiraceae, Propionibacterium and Weissella were found to be characteristic of uninfected animals (FDR?<?0.05), while Lactobacillus and Candidatus Arthromitus were characteristic of infected animals (FDR?<?0.05). Both these taxa have been reported as immunostimulatory, involved in immunological disorders. In infected animals the inferred metagenome assessed by PICRUST clearly showed a positive correlation between the presence of M. ornithogaster and KEGG genes related to ether lipid metabolism, already reported to be immunostimulatory by activation of macrophages and to play a pathophysiological role in several immunological disorders. Finally, our results show an interaction between infection of the digestive tract and intestinal microbiota of pet birds and provide insight into the changing of the complex enteric bacterial community.

• HIGHLIGHTS

• Macrorabdus ornithogaster is a gastric yeast that colonizes a wide range of birds.

• Differences were found between infected and healthy animals in gut microbiota.

• Candidatus Arthromitus was closely associated with infected birds.

• M. ornithogaster can affect intestinal microbiota composition of canaries.

     

Antihistomonal effects of artemisinin and Artemisia annua extracts in vitro could not be confirmed by in vivo experiments in turkeys and chickens

Five different Artemisia annua-derived materials (i.e. dry leaves, pure artemisinin, and hexane, dichloromethane or methanol extracts of leaves) were screened for their in vitro activities against six clonal cultures of Histomonas meleagridis. Except for the methanol extract, all tested materials displayed in vitro activity against all tested protozoal clones. Neither the dry plant material, extracts nor artemisinin showed any antibacterial activity against the xenic bacteria accompanying the six H. meleagridis clones at concentration levels identical to the antihistomonal setting. The dichloromethane extract of dry leaves (Ext-DCM) (minimal lethal concentration=1.0 mg/ml) and artemisinin (half-maximal inhibitory concentration=1.295 mg/ml) had the most promising antihistomonal properties and were therefore subsequently tested in a standardized experimental infection model in both turkeys and chickens infected with clonal H. meleagridis. There were no differences between treatment groups, where all infected turkeys showed severe clinical histomonosis and demonstrated severe typhlohepatitis typical for histomonosis. Consistent with the infection model used, the infected chickens did not show any adverse clinical signs but contracted severe lesions in their caeca 7 and 10 days post infection (d.p.i.), liver lesions were absent to mild after 7 d.p.i. and progressed to severe lesions at 10 d.p.i.; thus no differences between treatment groups were observed. In conclusion, neither artemisinin nor Ext-DCM was able to prevent experimental histomonosis in turkeys and chickens at the given concentrations, which is contrary to the antihistomonal effect noticed in vitro even though the same clonal culture was used. The results of this study therefore clearly demonstrate the importance of defined in vivo experimentation in order to assess and verify in vitro results.     

Molecular identification and phylogenetic relationships of trichomonad isolates of galliform birds inferred from nuclear small subunit rRNA gene sequences

Histomonas meleagridis is the etiological agent of histomonosis or blackhead disease. Recently, genotyping, based on polymerase chain reaction and sequencing of internal transcribed spacer-1 sequences was applied to various isolates originating from fowl. Three genotypes were described: types I and II isolates were associated with clinical disease and probably derived from H. meleagridis, whereas, type III isolates were not disease-associated and likely corresponded to Parahistomonas wenrichi according to morphological observations. However, this latter species has never been characterized at the molecular level and its phylogenetic relationships with other parabasalids remained hypothetical. To confirm the identification of these isolates, small subunit rRNA gene sequences were obtained from representatives of types I, II, and III and analyzed in a broad phylogeny including 64 other parabasalid sequences. From our phylogenetic trees, we confirmed that types I and II isolates were closely related, if not identical, to H. meleagridis, while type III isolates represented P. wenrichi. Both species clustered together with high support. This grouping suggested that speciation leading to these two species inhabiting the same hosts and ecological niche occurred recently in birds. In addition, speciation was likely followed by loss of pathogenicity in P. wenrichi.     

Histomonas meleagridis : Immunological Responses to • in the Turkey and Fowl

The immune responses of the turkey and fowl to intra-rectal infections with tissue containing Histomonas meleagridis were studied.

A protective immunity was produced in drug-treated turkeys and in fowls recovering spontaneously. These birds develop precipitating antibodies in their sera to an antigen derived from H. meleagridis. Antigen was first detected in caecal contents 4 days after infection and serum precipitins 7 days later. It was not possible to transfer protective immunity by injections of serum from immune to susceptible birds.

     

Protection afforded by avian influenza vaccination programmes consisting of a novel RNA particle and an inactivated avian influenza vaccine against a highly pathogenic avian influenza virus challenge in layer chickens up to 18 weeks post-vaccination

The efficacies of an oil adjuvanted-inactivated reverse genetics-derived H5 avian influenza virus (AIV) vaccine and an alphavirus replicon RNA particle (RP) AIV vaccine were evaluated in commercial Leghorn chickens. Challenge utilized A/turkey/MN/12582/2015, an isolate representing the U.S. H5N2 Clade 2.3.4.4 responsible for the 2015 highly pathogenic avian influenza (HPAI) epornitic in commercial poultry the United States. As part of a long-term, 36-week study, chickens were challenged at seven weeks of age after receiving a single vaccination, at 18 weeks of age following a vaccine prime-single boost, and at 36 weeks of age after a prime- double-boost. All vaccine programmes reduced virus oropharyngeal and cloacal shedding and mortality compared to the non-vaccinated control birds; however, chickens receiving at least one administration of the RP vaccine generally had diminished viral shedding especially from the cloacal swabbings. A detectable serum antibody response and protection were observed through 18 weeks post-vaccination. Our data suggest that, in conjunction with a comprehensive eradication, enhanced biosecurity and controlled marketing plan, vaccination programmes of commercial layer chickens with novel RP vaccines may represent an important tool for preventing HPAI-related mortalities and decreasing viral load during a catastrophic influenza outbreak.

RESEARCH HIGHLIGHTS

• Immunization of poultry following a vaccination schedule consisting of inactivated and RNA particle vaccines offered significant protection against lethal disease following HPAIV challenge.

• Virus shedding was significantly (P?<?0.05) reduced in chickens vaccinated with either inactivated and/or recombinant vaccines.

• Serum antibody titres were not a reliable indicator of protection.

• An inactivated vaccine containing 384 HAU of the homologous antigen was unable to induce complete protection.

     

Fatal necrotic tracheitis by Aviadenovirus in captive Alagoas curassows (Pauxi mitu) extinct from the wild

Extinct from nature, captive young Alagoas curassows (Pauxi mitu) were found agonizing or dead with respiratory disease. Intranuclear inclusion bodies were found in the epithelia of the trachea, associated with marked necrotic tracheitis. An Aviadenovirus was isolated in chicken eggs and characterized genetically with 99% identity to the fowl Aviadenovirus A, as based on the hexon protein gene. This is the first report of respiratory disease caused by Aviadenovirus in any cracid species in Brazil, recommending for stricter biosecurity in the conservation premises.

RESEARCH HIGHLIGHTS

• Fatal tracheitis in curassows extinct from nature was associated with Aviadenovirus A.

• Seven-month-old Alagoas curassows (Aves: Cracidae) died with haemorrhagic tracheitis.

• Aviadenovirus A with 99% identity to fowl adenovirus 1 was detected in dead curassows.

• Fatal tracheitis by Aviadenovirus was described in Pauxi mitu (Aves: Cracidae).

     

Genotyping of Riemerella anatipestifer by ERIC-PCR and correlation with serotypes

Riemerella anatipestifer (RA) is a widely distributed bacterial pathogen of birds responsible for remarkable losses to poultry production, especially among waterfowl. We characterized the genomic diversity of 166 field isolates of RA, collected from geese and ducks, using enterobacterial repetitive intergenic consensus (ERIC)-polymerase chain reaction (PCR). The field strains and five reference strains showed 17 distinct patterns consisting of five to 12 bands ranging from approximately 150–1800bp. The majority of the strains belonged to two closely related ERIC-PCR types (A and B), while the other types contained only a few isolates each. There was no association between ERIC-PCR type and host species, place, or year of isolation; however the ERIC-PCR pattern was correlated with serotype for most isolates. The majority of serotype 1 strains (101/107) belonged to ERIC-PCR type A while the remaining six strains represented five different ERIC-PCR types (D, G, L, M, and O). Serotypes 1,7 and 7 corresponded to ERIC-PCR types B and C, respectively. Serotypes 2, 4, and 10 could be subdivided by ERIC-PCR revealing two to four patterns within each serotype. These results indicate that ERIC-PCR may be a suitable technique for the molecular identification of RA serotypes, and the detection of subtypes within certain serotypes may aid further epidemiological investigations.

RESEARCH HIGHLIGHTS

• ERIC-PCR analysis of field R. anatipestifer strains revealed 17 distinct patterns

• Most strains belonged to two closely related ERIC-PCR types

• Serotype 1 was the most prevalent serotype representing 64.5% of the strains

• ERIC-PCR may be suitable for molecular identification of R. anatipestifer serotypes

     

Susceptibility of different turkey lines to <Emphasis Type="Italic">Histomonas meleagridis</Emphasis> after experimental infection

In the present investigation, three turkey lines, namely wild Canadian turkeys (WCT), British United turkey (BUT-Big6) and Kelly–Bronze turkeys (KBT) were compared for their susceptibility to infection with Histomonas meleagridis. All birds were kept on wood shaving as litter from day 1 on during the entire observation period. On day 28, 18–20 birds per turkey line were infected with H. meleagridis intracloacally. All birds were observed for 4 weeks after infection. The mortality rate was 95% in WCT, 78% in BUT-Big6 and 75% in KBT. In WCT, the first deaths occurred at day 6 and ended at day 13 post-infection, whilst for BUT-Big6 and KBT, birds died from days 10 to day 20. In KBT group, the mortality started at day 10 and lasted until day 17 after infection. At necropsy, all birds that died showed lesions typical for histomoniasis in the caeca and liver. The obtained results demonstrate that all tested turkey lines are susceptible to infection; however, the mortality rate for the wild Canadian turkey is statistically significantly higher compared to the other tested two lines.     

Anatomopathological changes,quantification and distribution of Libyostrongylus spp. in regions of the proventriculus and ventriculus of naturally- and experimentally-infected ostriches

Nematodes of the genus Libyostrongylus parasitize ostriches, causing high mortality rates. These nematodes are found in the proventriculus and ventriculus of ostriches, but little is known about their distribution and the possible anatomopathological changes they cause in the various regions of these organs. This paper describes the distribution and quantification of Libyostrongylus and pathological changes found in regions of the proventriculus and ventriculus of ostriches with high and low levels of both natural and experimental infection. Ostriches were necropsied and tissue samples from the distinct regions of both organs were analysed based on nematode counts and histopathology after staining with haematoxylin and eosin, Masson’s trichrome or Alcian blue/PAS. The cranial and glandular regions of the proventriculus were the most parasitized. The ventriculus contained more nematodes in the caudal region. No macro- or microscopic pathological changes were observed in either of these organs of experimentally-infected birds. However, naturally-infected birds with high levels of infection presented proventriculus with macroscopic lesions and heterophilic infiltrates surrounding nematodes. In the glandular region of this organ, nematodes were located in the adenomeres of the secretory ducts, causing altered architecture and erosions and ulcerative lesions with damaged epithelium. Nematode eggs were found in the koilin layer of the middle and caudal regions of the ventriculus only of these birds. The pH of the regions assessed by Alcian blue/PAS staining changed from acidic in the proventriculus to more alkaline in the caudal region of the ventriculus. These data add knowledge to the biology of Libyostrongylus.

RESEARCH HIGHLIGHTS

• The most parasitized areas were the cranial and glandular regions of the proventriculus.

• Naturally-infected birds with high levels of infection presented macro lesions in the proventriculus and damaged epithelium.

• Nematode eggs were found in the ventriculus.

• The proventriculus had an acidic pH, which turned alkaline towards the ventriculus.