Evaluation of bacterial shedding by budgerigars inoculated with Salmonella enterica serotype Saintpaul
(Avaliação da disseminação bacteriana por periquitos inoculados com Salmonella enterica sorotipo Saintpaul)
Elisângela de Souza Lopes1*, William Cardoso Maciel1, Débora Nishi Machado1, Átilla Holanda de Albuquerque1, Windleyanne Gonçalves Amorim Bezerra1, Ruben Horn Vasconcelos1, Raul Antunes Silva Siqueira2, Temístocles Soares de Oliveira Neto2, Ricardo Barbosa de Lucena2, Antônio Jackson Forte Beleza, Bruno Pessoa Lima1, Isaac Neto Goes da
Silva4, Rosa Patrícia Ramos Salles3, Régis Siqueira de Castro Teixeira1
Submetido: Avian Pathology
Evaluation of bacterial shedding by budgerigars inoculated with Salmonella Saintpaul
Elisângela de Souza Lopes1*, William Cardoso Maciel1, Débora Nishi Machado1, Átilla Holanda de Albuquerque1, Windleyanne Gonçalves Amorim Bezerra1, Ruben Horn Vasconcelos1, Raul Antunes Silva Siqueira2, Temístocles Soares de Oliveira Neto2, Ricardo Barbosa de Lucena2, Antônio Jackson Forte Beleza, Bruno Pessoa Lima1, Isaac Neto Goes da
Silva4, Rosa Patrícia Ramos Salles3, Régis Siqueira de Castro Teixeira1
1Laboratory of Ornithological Studies (LABEO). Postgraduate Program in Veterinary Science (PPGCV), Veterinary Faculty, State University of Ceará (UECE), Itaperi Campus, Fortaleza, Ceará, Brazil.
2Animal pathology Laboratory. Veterinary Science Department, Federal University of Paraíba (UFPB). Areia Campus, Areia, Paraíba, Brazil.
3Clinical Laboratory Pathology – BIOLAB
4Clinical Pathology Laboratory. Veterinary Faculty, State University of Ceará (UECE), Itaperi Campus, Fortaleza, Ceará, Brazil
* Corresponding author: [email protected]
Abstract
This study aimed to evaluate shedding, invasive ability and histopathological alterations of budgerigars (Melopsittacus undulatus) inoculated with a Salmonella Saintpaul strain. Two inoculi of 2x105 (I5) and 2x106 (I6) were prepared with Salmonella Saintpaul and administered via gavage to two groups of 22 adult budgerigars. Microbiological analyses to detect the pathogen in faeces and cloacal swabs were performed during 14 days post-inoculation (dpi), in addition to histopathological examinations in 3, 7 and 14 dpi. During the experiment, there was no mortality and a single bird presented typical clinical signs of salmonellosis. All cloacal swab samples were negative and a faecal sample from one bird in I5 group was positive in the first dpi. In addition, most organ samples were negative and only some birds that received the higher inoculum (I5) presented positive results. In 3 dpi, intestinal samples were positive, while in 7 dpi two birds presented positive spleen, intestine and liver samples. However, there were microscopic findings in all of the investigated samples. Although clinical signs and bacterial shedding were rare or absent, the presence of macroscopic and microscopic alterations indicate that Salmonella Saintpaul was capable of causing infection and lesions in organs of budgerigars that received two different oral doses of the bacterial inoculum.
Keywords: Salmonella Saintpaul, Psittaciformes, histopathological lesions, pathogenesis, bacterial concentration, clinical signs and bacterial shedding.
Introduction
Studies with Salmonella enterica in poultry are common in the scientific literature, whether evaluating epidemiological or pathogenic aspects (Kanashiro et al., 2005; Marcq et al., 2011; Albuquerque et al., 2016). However, despite the fact that studies involving this pathogen and wild birds are not performed as often, in the last decades several reports with psittacine have demonstrated the isolation of important paratyphoid serotypes, such as Salmonella Typhimurium and Enteritidis (Marietto-Gonçalves et al., 2010; Hidasi et al., 2013). In addition, information on the pathogenesis of salmonellosis in these birds is practically inexistent, especially with less frequent serotypes.
Among several serotypes involved in foodborne infections, Salmonella Saintpaul is one of the most frequently associated with outbreaks of human salmonellosis (Munnoch et al., 2009; Mody et al., 2011). However, this serotype have been reported in other studies with animals, including wild birds and poultry (Osman et al., 2010). In addition, this pathogen have been reported in clinically healthy psittacine from a commercial breeder (Ara chloropterus) and from illegal wildlife trade (Amazonas amazonica), both in Ceará State, Brazil (Lopes et al., 2014; Lopes et al., 2015).
Currently, there are no studies assessing pathogenicity of Salmonella Saintpaul in psittacine, unlike serotype Typhimurium, which several reports indicate that is the most frequent and virulent salmonella serotype in these birds (Vigo et al., 2009, Piccirillo et al., 2010).
Due to the lack of information, concerning pathogenic aspects of paratyphoid serotypes in psittacine, this study aimed to evaluate shedding, invasive ability and histopathological alterations of budgerigars inoculated with Salmonella Saintpaul.
Material and Methods
Birds. The experiment was performed in the inoculation facilities of the Ornithological Sector in the State University of Ceará (UECE). Budgerigars (Melopsittacus undulatus) aged six to twelve months were used. Birds were maintained in galvanized wire cages (22x21x16cm) in pairs (male and female). During the entire experiment, temperature (±20°C) and light hours (12 hours/day) were controlled and all birds received sterile water and feed ad libitum. This study was approved by the local Ethics Committee for the use of Animals of the State University of Ceará with the following protocol number: 127697942.
Pre-inoculation evaluation. All birds used in this study were initially submitted to physical examination to detect and discard unfit individuals. Haematological analysis was used to assess health status and results were compared to reference values for the species.
Approximately 0.3mL of blood was collected from the right jugular vein and placed in tubes with EDTA (Fischer et al., 2006).
To guarantee the absence of Salmonella spp., microbiological procedure was performed in samples from all birds according to the methodology used by Zancan et al.
(2000) with modifications. Briefly, individual cloacal swab samples were collected and placed in tubes containing selenite-cystine broth with novobiocin (SCNov) (40µg/mL). After incubation, samples were inoculated in plates with brilliant green agar (BGA) and incubated.
Then, colonies with morphological characteristics of Salmonella spp. were identified through biochemical methods and, if positive, birds were removed from the experiment.
Inoculum preparation. A strain of Salmonella Saintpaul resistant to nalidixic acid (SSNalr) isolated from a parrot (Amazona amazonica) in a local Wildlife Rehabilitation Center in Fortaleza, Brazil, was used to prepare the inoculum. This strain belonged to the collection of the Laboratory of Ornithological Studies (LABEO) and was isolated in a previous study
(Lopes et al., 2015). The inoculum was prepared according to the methodology by Berchieri Jr. et al. (2001), with some modifications as follows: strain was reactivated in 10mL buffered peptone water 0.1% and incubated statically. Then, serial dilutions were performed to count the colony forming units (CFU) (Miles et al., 1938). With these results, two inocula were prepared with saline solution: I5 (2x105 CFU/0.7mL of SSNalr) and I6 (2x106 CFU/0.7mL of SSNalr).
Experimental design. A scheme to illustrate the methodology is displayed in Figure 1.
During the experiment, two aspects were evaluated: 1- SSNalr shedding in faeces and swabs during post inoculation period; 2- Bacterial invasion performed by SSNalr in organs throughout the experiment.
Figure1.Dsitribution scheme of budgerigar groups inoculated with Salmonella Saintpaul.
Legend: Aspect 1. E5 (ten birds received inoculum I5); E6 (ten birds received inoculum I6); EC (control group, composed by six birds); Aspect 2: CG (six birds euthanized in pre-inoculation period); D3-I5 (five birds inoculated with I5 and euthanized in 3dpi); D3-I6 (five birds inoculated with I6 and euthanized in 3dpi); D7-I5 (five birds inoculated with I5 and euthanized in 7dpi); D7-I6 (five birds inoculated with I6 and euthanized in 7dpi); D14-I5 (ten birds inoculated with I5); D14-I6 (ten birds inoculated with I6); D14-C (six birds not inoculated).
To evaluate aspect 1, a total of 26 birds were used and divided in three groups, which received 0.7mL of inocula with the following distribution: E5 (ten birds received inoculum I5); E6 (ten birds received inoculum I6); EC (control group, composed by six birds). Every 24h for a period of 14 days post inoculation (dpi), individual cloacal swab and faecal samples were
collected for microbiological monitoring. The exact moment before the inoculation was defined as 0 dpi.
In order to evaluate aspect 2, additional 26 birds were distributed in the following groups: CG (six birds euthanized in pre-inoculation period); D3-I5 (five birds inoculated with I5 and euthanized in 3dpi); D3-I6 (five birds inoculated with I6 and euthanized in 3dpi); D7-I5 (five birds inoculated with D7-I5 and euthanized in 7dpi); D7-I6 (five birds inoculated with I6 and euthanized in 7dpi). In addition, all birds that survived the evaluation of aspect 1 were euthanized in 14 dpi, which was the end of the experiment. These were then renamed as the following groups: D14-I5 (ten birds inoculated with I5); D14-I6 (ten birds inoculated with I6); D14-C (six birds not inoculated).
Birds were euthanized with ketamine overdose administered via IV injection in the jugular vein. Then, necropsy was performed aseptically, during which fragments of liver, spleen and intestine were collected for microbiological and histological examination.
Microbiological procedure. Cloacal swab samples were processed according to the methodology of Zancan et al (2000) with some modifications described previously. Faecal samples were pooled and organ fragments were macerated individually, placed in tubes containing SCNov and incubated. Then, aliquots were streaked in plates containing brilliant green agar supplemented with nalidixic acid (100µg/mL) (VBNalr). After incubation, colonies with morphological characteristics of Salmonella Saintpaul (Koneman et al., 2012) were confirmed with rapid slide agglutination test using polyvalent antiserum O (PROBAC).
Histopathological procedure. Birds from groups CG, D3-I5, D3-I6, D7-I5, D7-I6, D14-I5 and D14-I6 were submitted to euthanasia and necropsy was performed to collect samples and evaluate macroscopic alterations. Fragments from liver, spleen and intestine were collected from each bird, pooled in five per group and fixated in 10% formalin for histopathological
analysis. Then, samples were submitted to standard histopathological procedure, using paraffin and slides were mounted with 5μm slices stained with haematoxylin-eosin, analysed with light microscope. Histopathological analysis was performed in the Laboratory of Histopathology in the Federal University of Paraíba (UFPB), Areia Campus.
Results
Clinical signs. During the experiment, no bird died in any group, while a single bird from I5 presented clinical signs compatible with salmonellosis, which were ruffled feathers (3dpi) and diarrhoea (14dpi).
Microbiological examination of swabs and faeces. During the experiment, Salmonella was not isolated in any cloacal swab samples from the evaluated groups (E5, E6 and EC).
However, one faecal sample was positive in 1 dpi from birds of group E5.
Microbiological examination of organs. A low rate of SSNalr recovery in the investigated organs was observed, which occurred only in birds from the higher inoculum group (I6).
Intestine from one bird was positive in 3dpi. On 7 dpi, two birds presented positive spleen, intestine and liver. All other samples were negative.
Macroscopic findings. Non-inoculated budgerigars submitted to necropsy in 0 and 14 dpi did not present any gross alterations. Several findings were observed in birds from both groups in different days of investigation (Table 1). However, some alterations were more frequent in 7dpi, which were hepatomegaly (80%) and splenomegaly (60%) in birds from D7-I6 and hepatic congestion (60%) in birds from D7-I5. Considering gross alterations of the entire experiment, splenomegaly was the most frequent (30%), followed by hepatic congestion (22.5%), hepatomegaly (17.5%), hepatic haemorrhage (17.5%) and pulmonary congestion (12.5%) (Figure 2).
Table 1. Absolute and relative frequency of gross alterations observed in budgerigars inoculated with SS
- Non-inoculated birds submitted to necropsy in 0 and 14 dpi were negative and results were not displayed in the table.
* Percentage of lesions detected in 14dpi was based in 10 birds, while in the remaining rates were calculated for 5 in the remaining days of observation.
Figure 2. Total percentage of most frequent gross alterations in budgerigars inoculated with Salmonella Saintpaul observed in 3, 7 and 14 days post-inoculation.
Histopathological alterations: Control group. Birds from the control groups did not present any alteration indicating Salmonella infection or any other disease.
I5 groups. Liver necrosis and inflammation were the most frequent lesions in birds from I5 groups in 3, 7 and 14 dpi. All organ samples from each day of necrosis in the same experimental group presented similar histopathological alterations. In 3 and 7 dpi, birds presented random necrosis of hepatocytes, associated with inflammation composed by heterophils, lymphocytes and plasma cells. In 3dpi, hepatic sinusoids were diffusely congested. In addition, intense diffuse congestion was observed in 14 dpi. Intestine samples in 3 dpi revealed inflammation in lamina propria and in 14 dpi moderate diffuse congestion, with lymphoid aggregates infiltrated by macrophages and heterophils. Spleen samples in 14 dpi presented macrophage infiltration within the parenchyma resulting in loss of the normal architecture of the organ.
I6 groups. Similar to what occurred in I5 groups, birds from I6 presented necrosis and inflammation in liver samples. In addition, in each day of necrosis, all slides presented similar findings. Lesions in liver were more intense in 3 dpi, in which samples presented intense diffuse congestion and multiple inflammation foci composed by histiocytes and epithelioid macrophages, in addition to a low number of lymphocytes and plasma cells. Cytoplasm of some macrophages and Kupffer cells presented a yellowish brown pigment (interpreted as hemosiderin) (Figure 3). In 14 dpi, random and periportal inflammation composed by lymphocytes and plasma cells was observed, associated with proliferation of bile ducts.
Intestine samples in 3 dpi presented an intense infiltration of mixed inflammatory cells (heterophils, macrophages, lymphocytes and plasma cells). In addition, loss of enterocytes associated with luminal haemorrhage was observed. In 7 dpi, lamina propria of the intestine
was diffusely infiltrated by macrophages, plasma cells and some heterophils. However, in 14 dpi, intense diffuse congestion was observed, in addition to a mixed inflammation in the lamina propria associated with loss of intestinal crypts. Spleen samples in 3 dpi presented intense diffuse congestion, thick capsule and infiltrate composed by macrophages and heterophils. In addition, in 14 dpi macrophage infiltration in the parenchyma was observed.
A
D B
C
Figure 3. Experimental infection of Salmonella Saintpaul in budgerigars. A: Liver (group E6 in 3dpi) – multiple inflammatory foci randomly distributed associated with the presence of a yellowish brown pigment (hemosiderin) in the cytoplasm of histiocytes and Kupffer cells (arrows). HE, obj. 40x. B: Liver (group E6 14dpi) – Periportal inflammation composed by numerous histiocytes, epithelioid macrophages and some heterophils, lymphocytes and plasma cells (arrows) associated with proliferation of bile ducts (arrowhead). HE, obj. 40x. C: Spleen (group E5 14dpi) – infiltration of macrophages in parenchyma, resulting in loss of normal architecture of the organ. HE, obj. 40x. D: Intestine (group E6 3dpi): intense inflammatory infiltration, composed by heterophils, macrophages, lymphocytes and plasma cells in the lamina propria (arrows). HE, obj. 20x.
Discussion
The absence of mortality and occurrence of clinical signs in a single budgerigar is not enough to affirm that Salmonella Saintpaul is not pathogenic for birds. A study performed by Andel et al. (2015) resulted in mortality and clinical signs compatible with salmonelosis, such as ruffled feathers, depression and poor body score condition in adult free-living passerines in the island Tititiri Matangi, New Zealand, associated with the presence of Salmonella Saintpaul. Studies have demonstrated that the inoculating dose of Salmonella in birds determines the occurrence of mortality and manifestation of clinical signs typical of salmonellosis (Noguera et al., 1992; Connolly et al., 2006; Rocha-e-Silva et al., 2013). In this context, experimentally infected free-living sparrows inoculated with Salmonella Typhimurium in a dose of 105 CFU presented mortality and clinical signs typical of salmonellosis in two birds out of six. However, an inoculum of 108 CFU caused mortality in all birds, in addition to an increased occurrence of clinical signs (Connolly et al., 2006).
Another important factor that may have influenced the results in this study and may explain the absence of mortality or clinical signs is the age. Budgerigars used in this study were adults and in this phase when infected by paratyphoid salmonella, usually there is a responsive immune system. In addition, the microbiota of adult birds is responsible for protecting against Salmonella serotypes (Gast, 2008).
The concentration of Salmonella Saintpaul may have hindered the recovery of this microorganism in swabs and faeces, considering that when an insufficient inoculating dose is used, bacterial excretion may not be detected in cloacal swab samples (Borsoi et al., 2011). In addition, serotype and species may also influence the results in inoculation experiments.
Albuquerque et al. (2016) infected orally one-day-old chicks with Salmonella Typhimurium using doses similar to this study. These authors verified that in 1, 4, 7 and 14 dpi the
frequency of birds shedding the pathogen in faeces and swabs was elevated, which was detected at a minimum rate of 60% of birds in each day of observation.
The low isolation rate in faeces and absence in cloacal swab samples in budgerigars may also be explained by the fact that Salmonella is a facultative intracellular bacterium.
Therefore, this pathogen may infect the host without necessarily be eliminated in faeces due to individual or nutritional aspects, especially when birds are submitted to adequate sanitary and welfare conditions (Dlugosz et al., 2015).
Birds are less susceptible to the infection by Salmonella in the adult age (Beal et al., 2004) and there is not a detailed report in the scientific literature of intestinal or visceral organ colonization by Salmonella in experimentally infected psittacine. Unlike what occurs with poultry, in which studies demonstrate that in the first weeks of age, there is a development in the immune system within the intestine that interfere with the colonization and persistence of the inoculated pathogen (Marcq et al., 2011).
Budgerigars submitted to the more elevated bacterial dose presented low frequency of positive results in the organs. Although the dose may have influenced the results, it is important to emphasize that the bacterial isolation from extra-intestinal organs samples may be related to the virulence of the inoculated strain. The persistence of Salmonella in the enteric environment also depends on the virulence of the serotype, considering that the pathogen may penetrate the intestinal barrier, invade phagocytes and replicate, migrating then through the defence cells to organs of the reticuloendothelial system, such as liver and spleen, possibly developing septicaemia (Ohl and Miller, 2001).
Despite the absence or low frequency of clinical signs, bacterial excretion and isolation from organs, gross and microscopic alterations demonstrate that Saintpaul serotype was capable of causing infection and lesions in the birds. According to Sousa et al. (2013),
bacterial isolation from liver and spleen suggests that the pathogen inoculated orally passed through the blood stream to reach the organs. Therefore, this may explain why the gross alterations observed were concentrated on these organs. Macroscopic findings detected in budgerigars in this study were compatible with typical cases of Salmonella infection, which have been reported in naturally infected psittacine bactéria (Orosz et al., 1992; Ward et al., 2003; Vigo et al., 2009; Piccirillo et al., 2010).
In general, microscopic alterations observed in spleen, intestine and liver samples were similar to what have been described in the literature with psittacine infected by Salmonella Typhimurium or Gallinarum that died with or without clinical signs (Ward et al., 2003; Piccirillo et al., 2010; Tunca et al., 2013). Commonly reported findings were infiltration by heterophils, lymphocytes and macrophages; necrosis and inflammation in the intestine, spleen and liver; in addition to hemosiderosis. However, no mortality or relevant clinical signs were observed in this study. Therefore, the detected microscopic alterations may indicate only the invasive capacity of the inoculated strain and the immune response, which may have been capable of controlling the infection.
In conclusion, the oral doses of Salmonella Saintpaul used in this study inoculated in budgerigars did not cause in almost all birds clinical signs or bacterial shedding. However, important gross and microscopic alterations were observed in the evaluated organs, which demonstrates the invasive capacity of this strain and reveals pathogenic potential. Therefore, we suggest that a more elevated inoculating dose could lead to a more expressive occurrence of clinical signs and bacterial shedding.
Disclosure statement
The authors declare that they have no conflict of interests, affiliation or financial involvement in organizations with financial interest in the subject presented in this review.
Acknowledgements
The authors thank the Conselho Nacional Tecnológico e Desenvolvimento Científico (CNPq);
the Laboratório de Histopatologia, Federal University of Paraíba; and the Laboratório de Estudos Ornitológicos, State University of Ceará.
References
Andel, M.V., Jackson, B.H., Midwinter, A.C., Alley, M.R., Ewen, J.G., McInnes, K., Jakob, R.H., Reynolds, A.D. e French, N. (2015). Investigation of mortalities ssociated with Salmonella spp. infection in wildlife on Tiritiri atangi Island in the Hauraki Gulf of New Zealand. New Zealand Veterinary Journal, 63, 235–239.
Albuquerque, Á.H., Teixeira, R.S.C., Machado, D.N., Lopes, E.S., Horn, R.V., Lima, B.P., Marques, A.R., Silva, I.N.G., Salles, R.P.R. e Cardoso, W.M. (2016).
Semina: Ciências Agrárias, Londrina, 37, 1919-1928.
Barrow, P.A., Huggins, M.B., Lovell, M.A. e Simpson, J.M. (1987). Observations on the pathogenesis of experimental Salmonella Typhimurium infection in chickens.
Veterinary Science, 42, 94-199.
Beal, R.K., Powers, C., Wigley, P., Barrow, P.A. e Smith, A.L. (2004). Temporal dynamics of the cellular, humoral and cytokine responses in chickens during primary and secondary infection with Salmonella enterica serovar Typhimurium.
Avian Pathology, 33, p. 25-33.
Berchieri Jr. A., Murphy, C.K., Marston, K. e Barrow, P.A. (2001). Observations on the persistence and vertical transmission of Salmonella enterica serovars Pullorum and Gallinarum in chickens: effect of bacterial and host genetic background.
Berchieri Jr. A., Murphy, C.K., Marston, K. e Barrow, P.A. (2001). Observations on the persistence and vertical transmission of Salmonella enterica serovars Pullorum and Gallinarum in chickens: effect of bacterial and host genetic background.