Genetically different clonal isolates of Trichomonas gallinae, obtained from the same bird, can vary in their drug susceptibility, an in vitro evidence

Trichomonas gallinae is a flagellated protozoon which parasitizes in the upper digestive tract of different birds, especially columbiformes (doves and pigeons) and falconiformes. The parasite is also a common inhabitant of the crop of psittacine birds and is frequently detected in budgerigars. The lesions associated with T. gallinae infection of the upper digestive tract range from mild inflammation of the mucosa to large caseous lesions that block the lumen of the oesophagus. Nitroimidazoles are considered to be the drugs of choice for the treatment of trichomonosis. However, only a few studies report the existence of resistant strains of T. gallinae to these drugs. Thus, in the present investigation cloned cultures of T. gallinae obtained from budgerigars and pigeons were analysed for the first time for their in vitro susceptibilities against four 5′-nitroimidazole derivates, including metronidazole, dimetridazole, ronidazole and ornidazole. Significantly different minimal lethal concentrations (MLCs) were observed for them against all four drugs. The lowest MLCs revealed the Trichomonas isolates obtained from two budgerigars, ranging from 2.0 ± 0.3 to 3.0 ± 0.7 μg/ml for metronidazole and dimetridazole, and from 2.0 ± 0.6 to 6.7 ± 1.7 μg/ml for ornidazole and ronidazole. Contrary to this, the highest MLCs were recorded for one Trichomonas isolate obtained from a pigeon, ranging from 83.3 ± 6.7 (for dimetridazole and ronidazole) to 103.3 ± 3.3 μg/ml (for metronidazole and ornidazole). The data obtained for the resistance testing were further compared with already available genetic data of the small subunit rRNA gene sequences and ITS-1, 5.8S rRNA and ITS-2 sequences, indicating a certain correlation between in vitro results and strain relationships.     

Challenge infection as a means of determining the rate of disease resistant Trichomonas gallinae-free birds in a population

Trichomonas gallinae-free pigeons and mourning doves were infected with the Jones' Barn strain of T. gallinae to determine the rate of disease resistant T. gallinae-free birds in each population. Although all birds became infected 88% of the pigeons were resistant to trichomoniasis while 82% of the mourning doves were resistant. It was concluded that these birds had been previously infected and spontaneously lost their trichomonad fauna while retaining their resistance to fatal infection.     

Pathogenicity of Tritrichomonas mobilensis: subcutaneous inoculation in mice

The standard "subcutaneous mouse assay" was used to investigate the inherent pathogenicity of Tritrichomonas mobilensis, an intestinal parasite of squirrel monkeys. C57B1/6 mice given subcutaneous bilateral inocula of T. mobilensis died by day 4 postinoculation with lesions too small to be measured. Control mice similarly inoculated with pathogenic and nonpathogenic strains of Trichomonas vaginalis survived the challenge and produced lesions on day 6 with mean volumes in agreement with previous reports. CD1 mice similarly inoculated with standard and double doses of trichomonads (T. mobilensis) again produced small lesions. CD1 mice inoculated at double dosage were moribund or dead on days 5 and 6, respectively, postinoculation. Necropsies were performed on dead and sacrificed mice. Tissues were obtained from internal organs for histology and culture. Unexpectedly, trichomonads were cultured from liver and lung of C57B1/6 mice at the standard level of inoculation and liver, lung, and spleen of CD1 mice at the higher level of inoculation. Although trichomonads are normally considered surface-dwelling noninvasive organisms, the penetration of trichomonads to deep tissues is not without precedent. Tritrichomonas foetus and Trichomonas gallinae are known to invade tissues of their respective hosts. Trichomonas vaginalis has been demonstrated in subepithelial areas of both the prostate gland and cervix of humans. The ability of several species of trichomonads to invade tissues and/or migrate to other sites in their hosts suggests a need for revision of the concept of trichomonads as strictly lumen or surface-dwelling parasites.     

Serum protein changes in immune and nonimmune pigeons infected with various strains of Trichomonas gallinae

Serum protein changes were studied in immune and nonimmune pigeons infected with three different strains of Trichomonas gallinae. Strain I (nonvirulent) produced no change in the relative concentration of serum components. Strains II (oral canker) and III (Jones' Barn) produced decreases in albumin and alpha globulins, and increases in beta and gamma globulins between the 7th and 20th days post infection. Birds infected with strain II began to return to normal by the 20th day, while all those infected with strain III were dead between 10 and 14 days post infection. Two serum protein patterns resulted from infection of immune birds with the Jones' Barn strain. One showed no change in relative protein concentrations and no tissue invasion by the parasite while the other was similar to that seen in nonimmune birds infected with a strain producing oral canker. These also showed evidence of tissue invasion by the parasite. It was concluded that tissue invasion was necessary to evoke a quantitative change in serum protein concentrations.     

Genetic differentiation and biochemical polymorphism among trichomonads

Isoenzyme electrophoresis was used to study levels of genetic differentiation among strains and clones of Trichomonas gallinae, Trichomonas vaginalis, Tritrichomonas foetus, Tetratrichomonas gallinarum, and Pentatrichomonas hominis. Strain variation was found within T. gallinae, T. vaginalis, and T. foetus, however, levels of enzyme polymorphism were greater in T. gallinae than in T. vaginalis or T. foetus. Isoenzyme genotypes were not a stable property of T. gallinae clones cultivated in vitro. Retrospective studies of T. gallinae SG and JB6 clones revealed that mutation occurred during in vitro cultivation. Heterozygotes of hexokinase-1 and phosphoglucomutase displayed 2 allomorphs in equal dosage, indicating that trichomonads are diploid for these protein loci. Phenetic clustering of the biochemical data suggests that levels of genetic divergence among the species studied are extensive.     

The prevalence and intensity of external parasites in domestic pigeons (Columba livia domestica) in Egypt with special reference to the role of deltamethrin as insecticidal agent

This study aimed to investigate the prevalence and intensity of external parasites in domestic pigeons in Giza, Egypt, from January 2020 to December 2020. A total of 300 domestic pigeons (25 pigeons per month) were examined. The birds were divided into groups based on their age. The oxidative stress parameters; serum zinc concentration, serum malondialdehyde (MDA), and serum Nitric oxide were evaluated in single and mixed external parasitic infestations. The prevalence of external parasites in examined pigeons was 80.3%. The detected parasites were Pseudolynchia canariensis (P. canariensis), Hippobosca equina (H. equina), Columbicola columbae (C. columbae), Menopon gallinae (M. gallinae), Knemidocoptes species (spp.) and Dermanyssus gallinae (D. gallinae); their incidences were 41.6, 26, 7, 5,0.33 and 0.33%, respectively. The highest infestation was recorded in both spring and summer. . The incidence of disease was higher in squabs and young birds than in adults. The mixed external parasitic infestation was recorded in this study. The infected birds showed decreased serum zinc concentration and elevated MDA and serum Nitric oxide levels. In conclusion, regular monthly treatment with deltamethrin is recommended as an effective drug in the treatment of the infested birds and succeeded in reducing the incidence of externalparasites in the treated birds; in addition, pigeon management measures must be implemented to reduce the risk of external parasites.     

Trichomonas gallinae in columbiform birds from the Galapagos Islands

Domestic pigeons were introduced into the Galapagos Islands in 1972 or 1973. There is a high prevalence of Trichomonas gallinae among them and some evidence of canker. Trichomonas gallinae can be found also in endemic Galapagos doves in the vicinity of Puerto Ayora on Santa Cruz Island. Doves examined on pigeon-free islands were not found infected.     

Effect of virulent Trichomonas gallinae on the band-tailed pigeon.

Nine Trichomonas-free band-tailed pigeons (Columba fasciata fasciata) trapped in Colorado died from experimental infection with Jones' Barn Trichomonas gallinae 7.1 days (av.) post-inoculation. Three experimentally infected domestic pigeons (C. livia) used as controls died 5.7 days (av.) post-inoculation. Three band-tailed pigeons from Colorado naturally infected with avirulent T. gallinae. Trichomonads obtained from a fatal case of trichomoniasis in a band-tailed pigeon from California killed a band-tailed pigeon from Colorado; they did not kill five domestic pigeons but did induce severe oral caseation.     

Mouse spleen cell responses to trichomonal antigens in experimental Trichomonas vaginalis infection

Subcutaneous inoculation of live T. vaginalis into mice caused splenomegaly, particularly when using strains of parasites with low pathogenicity. The proliferative responses of spleen cells from uninfected mice, as measured by [3H] TdR uptake, showed that trichomonal antigens, whether from strains with high or low pathogenicity, have no mitogenic activity. Spleen cells from mice infected with trichomonads of low pathogenicity showed a proliferative response to trichomonal antigens that was maximal after 4 days incubation. The proliferative response of spleen cells from mice infected with trichomonads of high pathogenicity continued for at least 6 days in the presence of the antigen. Moreover, in the latter case there was a significantly greater uptake of [3H] TdR when cells were incubated with antigens of a highly pathogenic strain. These results support the view that although many antigens are common to strains with differing levels of pathogenicity, some antigens are more closely associated with strains that are more highly pathogenic. The strong proliferative response to these antigens may then be related to the clinical presentation of infection with these strains.     

Trichomonas gallinae in wintering Common Wood Pigeons Columba palumbus in Spain

We studied the prevalence and potential subclinical effects of infestation by Trichomonas gallinae in 91 hunter-harvested Common Wood Pigeons Columba palumbus from northern ( n  = 30) and southern ( n  = 61) Spain during the winter period. All animals were measured, sexed, aged, necropsied and their organs were weighed. Infestation with T. gallinae was diagnosed using three different methods: direct inspection for the presence of lesions, direct microscopic observation and culture. Of the sampled birds, 34.2% were positive for the presence of T. gallinae . Prevalence was significantly higher in adult Wood Pigeons than in the juvenile group, and prevalence was significantly lower in birds sampled from the north. No significant differences in prevalence were found between males and females. Culture was significantly more sensitive than the other methods of diagnosis. Parasitized birds were in poorer body condition, as revealed by their lower body mass and fat reserves. No significant variation could be detected in heart or spleen weight between parasitized and healthy birds. However, juvenile Wood Pigeons in which T. gallinae was detected had a significantly larger bursa of Fabricius. Variations in the prevalence of T. gallinae in Wood Pigeons could be related to migration as well as increased exposure through shared feed and water where these are artificially provided. We also discuss the potential effect of T. gallinae on body condition and the eventual risk for endangered predators through increased exposure to infected prey.     

Survival of Trichomonas gallinae in white-winged dove carcasses

Survival of Trichomonas gallinae was examined in white-winged dove (Zenaida asiatica) carcasses to assess whether birds that have been dead up to 8 hr can be sampled reliably for this protozoan. Carcasses of 100 T. gallinae-positive white-winged doves were separated into four groups of 25 birds, representing 2, 4, 6, and 8 hr post mortem sampling intervals and placed into an environmental chamber maintained at 27 C and 75% relative humidity. Live T. gallinae were isolated in 96, 100, 100, and 92% of the carcasses at each of the respective post mortem intervals. The experiment was repeated with another 100 carcasses of T. gallinae-positive white-winged doves placed in the environmental chamber, this time maintained at 27 C and 40% relative humidity. Live T. gallinae occurred in 96, 100, 96, and 100% of the carcasses at each of the respective post mortem intervals. Across both trials, the overall ability to detect positive birds from sampling carcasses up to 8 hrs post mortem was 97%. An a posteriori experiment was conducted in which 23 and 18 carcasses from the second trial were maintained in the environmental chamber at 27 C and 40% relative humidity and resampled at 24 and 48 hr post mortem, respectively. Live trichomonads were isolated from 91 and 44% of the carcasses at 24 and 48 hr, respectively. Results suggest live T. gallinae can be obtained from dove carcasses reliably up to 8 hr and possibly up to 24 hr after host death. The ability for T. gallinae to survive within this time interval can aid wildlife personnel in monitoring this protozoan at hunter check stations or obtaining samples from recently killed birds.     

Experimental transfer of Trichomonas gallinae (Rivolta, 1878) from white-winged doves to mourning doves

Isolates of Trichomonas gallinae (Rivolta, 1878) from white-winged doves, Zenaida asiatica (L.), were transferred experimentally to young mourning doves, Zenaida macroura (L.). Twenty-three of 25 mourning doves developed infections with isolates of T. gallinae from 25 white-winged doves. In addition, eight of eight rock doves (Columba livia Gmelin) were infected with duplicate isolates. All infected recipient birds harbored avirulent isolates except for one mourning dove which died from extensive oral lesions. However, repeated attempts using this isolate of T. gallinae to produce lesions in additional recipients were unsuccessful. Despite the findings of this study, it was suggested that future dove management strategies consider the possibility of disease outbreaks involving white-winged doves and susceptible populations of mourning doves.     

Agar Techniques for Colonizing and Cloning Trichomonads

SYNOPSIS. Agar media have been used in plates, Burri tubes, and sealed slide preparations to grow trichomonads as clonal colonies. Periodic observation of slide-culture colonies confirmed their origin from single organisms as clones. Axenic cultures of Trichomonas vaginalis (3 strains), T. gallinae (2 strains), T. gallinarum (1 strain), Tritrichomonas augusta (5 strains), Trit. foetus (4 strains), Trit. suis (?) (6 strains), Pentatrichomonas hominis (1 strain), and an undescribed porcine form were used successfully. Urinary and vaginal specimens with T. vaginalis were also plated directly from patients. In all trials, organisms were recovered from colonies for broth culture and re-plating.
Colonies of the two strains of T. gallinae showed constant differences in their growth under the same conditions. Plated T. vaginalis have shown zones of growth inhibition in tests against drug-impregnated discs.     

Extensive diversity of intestinal trichomonads of non-human primates

Despite the fact that the non-human primates are our closest relatives and represent a species-rich mammalian group, little is known about their intestinal protistan parasites/commensals. Particularly, the intestinal trichomonads represent a neglected part of the fauna of the primate digestive system. We have established 30 trichomonad strains isolated from feces of 11 primate species kept in 3 Czech zoos and performed an analysis of their SSU rDNA and ITS1-5·8S rDNA-ITS2. Our results showed that intestinal trichomonads are rather common among non-human primates. Molecular phylogenetic analysis showed that the strains are unexpectedly diversified, belonging to 8 or 9 distinct species. Interestingly, the vast majority of the strains from non-human primates belonged to the genus Tetratrichomonas while no member of this genus has been found in the human intestine so far. In addition, hominoid and non-hominoid primates differed in their intestinal trichomonads. Our results suggest that captive primates possibly may be infected by intestinal trichomonads of other vertebrates such as pigs, cattle, birds, tortoises and lizards.     

Trichomonad infection in endemic and introduced columbids in the Seychelles

Island endemic avifaunas face many threats, including the now well-documented impacts of pathogens. The impacts of pathogens on the endemic Seychelles avifauna, however, have been little studied. The protozoan parasite Trichomonas gallinae has been shown to reduce survival and reproductive success of the endemic Pink Pigeon Columba mayeri on the nearby island of Mauritius. I investigated trichomonad infection prevalence and pathogenicity in endemic Seychelles Blue Pigeons, Alectroenas pulcherrima, and two introduced species of columbid, the Madagascar Turtle-dove, Streptopelia picturata, and the Barred Ground Dove, Geopelia striata, on the Seychelles island of Mahé during September-October 2007. I asked whether: 1) trichomonad infections occur in these species; 2) prevalence varies among species; and 3) birds show any signs of pathogenicity consistent with tricho-monosis. I use the results to assess the potential threat of this pathogen to A. pulcherrima. All three species were infected with trichomonads, and the overall prevalence was 27.5%. Alectroenas pulcherrima had higher prevalence (47.1%) than the two introduced species combined (24.3%). No infected individuals showed any signs of disease. These findings suggest that trichomonad parasites should be considered as a potential disease threat to the A. pulcherrima population.     

Does avian malaria reduce fledging success: an experimental test of the selection hypothesis

Like many parasites, avian haematozoa are often found at lower infection intensities in older birds than young birds. One explanation, known as the “selection” hypothesis, is that infected young birds die before reaching adulthood, thus removing the highest infection intensities from the host population. We tested this hypothesis in the field by experimentally infecting nestling rock pigeons (Columba livia) with the malaria parasite Haemoproteus columbae. We compared the condition and fledging success of infected nestlings to that of uninfected controls. There was no significant difference in the body mass, fledging success, age at fledging, or post-fledging survival of experimental versus control birds. These results were unexpected, given that long-term studies of older pigeons have demonstrated chronic effects of H. columbae. We conclude that H. columbae has little impact on nestling pigeons, even when they are directly infected with the parasite. Our study provides no support for the selection hypothesis that older birds have lower parasite loads because parasites are removed from the population by infected nestlings dying. To our knowledge, this is the first study to test the impact of avian malaria using experimental inoculations under natural conditions.     

Antigenic Analysis of Virulent and Avirulent Strains of Trichomonas gallinae by Gel Diffusion Methods*

SYNOPSIS. Antigenic constitution of 5 Trichomonas gallinae strains and substrains was analyzed by gel diffusion technics. Fresh isolates of the very virulent JB and of an avirulent SG strain as well as avirulent substrains JBC and SGC, derived from JB and SG respectively by prolonged in vitro cultivation, were used in the experiments. An originally avirulent AG strain that was attenuated still further and lost its infectivity for pigeons during many years of serial transfers in nonliving media also was analyzed. Two major groups of antigens, A and B, were differentiated on the basis of precipitin line patterns formed in gel diffusion reactions involving the 5 strains and substrains and antisera prepared in rabbits against each of these trichomonad stocks. Group A was subdivided further into subgroups [A] and (A). JB, JBC, AG, and SGC trichomonads appeared to share all or nearly all antigens of both these subgroups, but AG strain contained some unique [A] and (A) antigens in addition to those which it had in common with the remaining 4 strains and substrains. Group B antigens were divided into 5 subgroups, B₁ to B₅. The complete B₁ antigenic complex was found in JB and JBC trichomonads and part of this complex was present also in SG strain and SGC substrain. In all instances, subgroup B₁ antigens stimulated production of specific antibodies in rabbits and combined with these antibodies present in immune sera. The complete B₂ antigenic complex was found only in JBC substrain. Some subgroup B₂ antigens were present also in JB trichomonads. Very few of these, however, were capable of stimulating antibody production in rabbits. The more numerous B₂ elements of JB strain that did not stimulate immunologic responses in rabbits, might be in the form of incomplete hapten-like antigens. All subgroup B₂ antigens found in JB strain represented only a portion of the B₂ complex associated with JBC substrain. Subgroup B₂ was characteristic of SG and SGC trichomonads, the latter substrain differing from the parental SG strain in the levels of both B₂ and B₁ antigens; these differences, however, were purely quantitative. JB strain reacted with some of subgroup B₃ antibodies present in SG and SGC antisera, but failed to stimulate antibody formation against any of these antigens in rabbits. The B₃ elements of JB trichomonads might be incomplete antigens. AG strain was characterized by having B₄ and B₅ antigenic complexes. The very small part of subgroup B₄, represented by a weak precipitin line in reactions between JB strain or JBC substrain and anti-AG serum, suggested the presence of some incomplete B₄ antigens in these trichomonads. Irrespective of whether freshly isolated avirulent strains or substrains attenuated by prolonged in vitro cultivation are examined by gel diffusion, such organisms are found richer in subgroup B antigens than the fully virulent JB trichomonads. All the results suggest that there may be a direct relationship between antigenic constitution and virulence of T. gallinae strains.     

Genetic Diversity of Newcastle Disease Virus in Wild Birds and Pigeons in West Africa

In West and Central Africa, virulent Newcastle disease virus (NDV) strains of the recently identified genotypes XIV, XVII, and XVIII are enzootic in poultry, representing a considerable threat to the sector. The increasing number of reports of virulent strains in wild birds at least in other parts of the world raised the question of a potential role of wild birds in the spread of virulent NDV in sub-Saharan Africa as well. We investigated 1,723 asymptomatic birds sampled at live-bird markets and sites important for wild-bird conservation in Nigeria and 19 sick or dead wild birds in Côte d''Ivoire for NDV class I and II. Typical avirulent wild-type genotype I strains were found in wild waterfowl in wetlands in northeastern Nigeria. They were unrelated to vaccine strains, and the involvement of inter- or intracontinental migratory birds in their circulation in the region is suggested. Phylogenetic analyses also revealed that genotype VI strains found in pigeons, including some putative new subgenotype VIh and VIi strains, were introduced on multiple separate occasions in Nigeria. A single virulent genotype XVIII strain was found in a dead wild bird in Côte d''Ivoire, probably as a result of spillover from sick poultry. In conclusion, screening of wild birds and pigeons for NDV revealed the presence a variety of virulent and avirulent strains in West Africa but did not provide strong evidence that wild birds play an important role in the spread of virulent strains in the region.     

Investigation of shiga toxin-producing Escherichia coli in avian species in India

AIMS: To investigate the presence or absence of shiga toxin-producing Escherichia coli (STEC) in avian species in India. METHODS AND RESULTS: Faecal samples originating from 500 chicken and 25 free flying pigeons were screened for the presence of E. coli. A total of 426 (chicken, 401; pigeons, 25) E. coli strains were isolated. Of 426 E. coli strains, 387 were grouped into 77 serogroups, while 70 and 59 strains were untypable and rough, respectively. All isolates were subjected to multiplex polymerase chain reaction (m-PCR) for the detection of stx(1), stx(2), eaeA, hlyA and saa genes. None of the E. coli strains studied showed the presence of stx(1), stx(2) or their variants and saa genes. Overall 11 (2.74%) and seven (1.74%) strains from chickens possessed eaeA and hlyA genes, respectively, while as only six (1.49%) strains from chickens possessed both eaeA and hlyA genes. O9, O8, O60 and O25 serogroups were most predominant of which there were 24 (5.63%), 23 (5.39%), 23 (5.39%) and 20 (4.69%) strains, respectively. None of the isolates from pigeons showed the presence of any of the virulence genes studied. CONCLUSIONS: STEC are absent in chickens and pigeons. However, further studies are required in this direction to confirm or contradict our findings. E. coli strains originating from birds are carrying a low percentage eaeA or hlyA genes. SIGNIFICANCE AND IMPACT OF THE STUDY: The present study is the first attempt to investigate STEC in chickens and free flying pigeons in India. The chickens and pigeons cannot be considered as important carrier of STEC in India.