Antimicrobial-free control of fowl typhoid in laying hens - case report

[Controle eficaz do tifo aviário em poedeiras, sem uso de antimicrobianos - relato de caso]

ABSTRACT

Salmonella Gallinarum (SG) is a Gram-negative bacterium responsible for causing fowl typhoid (FT), a septicemic disease that results in significant losses in poultry farming. Although antimicrobials reduce clinical signs and mortality, they are not effective against the infection, as clinical signs may recur, and birds may continue to shed the pathogen after treatment. The irrational use of these drugs is one of the main factors related to the emergence of resistant strains, raising concerns for public and animal health. This report describes the alternative control of FT in cage-free laying hens on a poultry farm in Minas Gerais, Brazil. Following a sudden increase in mortality, five birds were sent to the Avian Diseases Laboratory at the Federal University of Minas Gerais for examination. Bacterial isolation, biochemical tests, rapid serum agglutination and PCR were performed, confirming SG infection in two birds. To control the outbreak, the live attenuated 9R vaccine was used in combination with the removal of sick birds, resulting in a reduction in mortality and clinical signs. It was concluded that, in the face of an outbreak of FT, biosecurity measures combined with vaccination using the 9R strain and removal of sick birds are effective in disease control.

Keywords:
Salmonella Gallinarum; salmonellosis; sanitary management; 9R

RESUMO

Salmonella Gallinarum (SG) é uma bactéria Gram-negativa responsável por causar o tifo aviário, doença septicêmica que gera altos prejuízos na avicultura. Embora os antimicrobianos reduzam os sinais clínicos e a mortalidade, eles não são eficazes contra a infecção, visto que pode ocorrer recorrência dos sinais clínicos e as aves podem continuar a eliminar o patógeno após o tratamento. O uso irracional desses fármacos é um dos principais fatores relacionados ao surgimento de cepas bacterianas resistentes, gerando preocupações para a saúde pública e animal. Este relato descreve o controle alternativo de tifo aviário em galinhas poedeiras criadas livres de gaiolas, em uma propriedade no interior de Minas Gerais, Brasil. Após relato de aumento súbito na mortalidade, cinco aves foram encaminhadas ao Laboratório de Doenças das Aves da Universidade Federal de Minas Gerais para realizar exame clínico e necropsia. Foram realizados isolamento bacteriano, testes bioquímicos, soroaglutinação rápida e PCR, o que confirmou a infecção por SG em duas das aves. Para controlar o surto, utilizou-se a vacina viva atenuada 9R e eliminaram-se as aves doentes, o que resultou na diminuição da mortalidade. Conclui-se que, diante de um surto de tifo aviário, medidas de biosseguridade associadas à vacinação com a cepa 9R e à eliminação de aves com sinais clínicos são eficazes no controle da doença.

Palavras-chave:
Salmonella Gallinarum; salmonelose; manejo sanitário; 9R

INTRODUCTION

Salmonellosis is a disease caused by bacteria of the Salmonella genus. These are Gram-negative bacilli, facultative intracellular, anaerobic or facultative aerobic, non-sporulating, with the majority being motile. In poultry, they can induce three different diseases, fowl typhoid, caused by Salmonella enterica subspecies enterica serovar Gallinarum biovar Gallinarum (SG), pullorum disease, caused by Salmonella enterica subspecies enterica serovar Gallinarum biovar Pullorum (SP), and fowl paratyphoid, caused by other serovars within subspecies enterica. Fowl typhoid (FT) is a significant concern, capable of inflicting substantial losses upon the poultry industry. Moreover, when FT is diagnosed in breeder flocks, the elimination of infected flocks is a mandatory measure (Freitas Neto et al., 2020).

Fowl typhoid is an acute or chronic septicemic disease that mainly affects adult birds, although it can manifest in different age groups, resulting in mortality rates that, although variable, tend to be high (Barrow and Freitas Neto, 2011). Semi-heavy lineage layers may show clinical signs such as lethargy, prostration, reduced feed intake, decreased egg production, and diarrhea, while birds from light lineages are resistant and do not show clinical signs (Freitas Neto et al., 2007; Berchieri et al., 2010). Bird mortality observed in outbreaks can reach 40-80% of the flock, whereby chickens become ill and die within 7-14 days. This process can occur continuously in the batch, reaching levels higher than those mentioned (Freitas Neto et al., 2020). Birds that survive the outbreak may experience weight loss and inadequate development for laying and reproduction (Shivaprasad, 2000), resulting in economic losses due to poor flock performance.

The use of antimicrobials is not effective in eradicating the infection and may result in the recurrence of clinical signs after treatment (Celis-Estupiñan et al., 2017). Therefore, antimicrobial treatment should be considered as a last resort for controlling FT (Shivaprasad, 2000). Additionally, antimicrobial resistance is a growing concern, especially due to bacterial plasticity, which facilitates the transfer of antimicrobial resistance genes to other pathogens important for human health (Sun et al., 2021).

The 9R vaccine, composed of a rough strain, Salmonella Gallinarum 9R (SG9R), developed by Smith (1956), is effective in reducing mortality and colonization by SG in the organs of infected birds. In a study by Lee et al. (2007), it was observed that 95-100% of the unvaccinated birds showed reisolation of the bacteria from internal organs and the cecum, whereas in the vaccinated group, the rate was only 10-40%. Furthermore, the removal of sick birds of the infected flock and the proper disposal of carcasses are effective measures in disease control (Berchieri et al., 2000). Additionally, vector control, as well as cleaning and disinfection of the poultry house, vehicle and visitor control, water and feed quality, and the acquisition of birds from establishments with sanitary certification, are also important for FT control (Shivaprasad, 2000).

This study reports that the use of the 9R vaccine, along with the removal of dead and sick birds from the flock, was able to effectively control an outbreak of fowl typhoid diagnosed early in a flock of semi-heavy breed laying hens raised in a cage-free production system, without the need for antimicrobial use.

CASE DESCRIPTION

This study reports the occurrence of FT in an egg production farm operating on a cage-free system located in a country town of the state of Minas Gerais, Brazil. In this poultry farm, laying hens are housed in closed barns. This specific flock consisted of 800 birds of semi-heavy lineage, received and housed at the beginning of 2023 at 16 weeks of age. This farm acquires birds close to laying age, rather than day-old chicks. Egg laying began around the 18^th week, and from that point, production increased gradually.

At 26 weeks, a sudden increase in mortality rate was observed in the flock, with the registration of 8 (1.0%) dead birds on the first day. On the second day, mortality reached 14 (1,8%) birds, representing the highest rate recorded during the outbreak. Mortality remained high during the outbreak and normalized starting from day 20 (Fig. 1). The egg production was stable even on the day with the highest mortality.

Figure 1
Mortality rate over the observation period of the outbreak. (*) Represents the day when the first dose of the 9R vaccine was administered to the entire flock.

In this scenario, five hens were brought to the Avian Disease Laboratory, located in the Department of Preventive Veterinary Medicine at the School of Veterinary Medicine of the Federal University of Minas Gerais (UFMG). Two birds died during transport and were identified as 1 and 2. The surviving birds and were euthanized in a CO₂ chamber were identified as birds 3, 4, and 5. Upon clinical examination, two birds showed signs of lethargy, prostration and greenish diarrhea. The two deceased birds had feces adhered around the cloaca.

In the gross pathological examination, the significant findings were as follows: mild ascites (bird 5); severely enlarged and congested liver with distended gallbladder (bird 2); moderately enlarged liver with greenish-brown coloration, moderately hyperemic duodenal mucosa, liquefied and greenish intestinal content (bird 1); distended gallbladder and diffuse intense hemorrhagic spots on the jejunal and ileal mucosa (bird 5); moderate petechiae in the ileum (bird 4); severely enlarged and congested spleen (bird 1), mildly enlarged spleen (bird 3); reactive cecal tonsils (birds 1 and 2); opaque air sacs, indicative of airsacculitis (bird 1), and pale (birds 1 and 2) and congested (bird 2) lungs; hydropericardium (bird 1) and whitish areas in the heart (birds 1 and 2); atrophied ovary and involuted oviduct (birds 1 and 2); irregular and shriveled ovarian follicles (bird 5); enlarged kidneys (birds 1, 2, 3, 4, and 5) with whitish regions (birds 1 and 4).

Liver and spleen swabs were directly streaked onto brilliant green agar (BGA - OXOID Ltd., Basingstoke, Hampshire, England) and incubated at 37°C for 18 hours. After incubation, growth of pink colonies, non-lactose fermenters, suggestive of Salmonella spp., was observed. Subsequently, biochemical tests were performed from the suspected colonies. For this purpose, triple sugar iron agar (TSI - NEOGEN, Lansing, Minnesota, USA) and lysine iron agar (LIA - HiMedia Laboratories Pvt. Ltd., Dindhori, Nashik, India) were used. After inoculating the suspected colonies, they were incubated at 37°C for 18 hours. As a result, in TSI, an acidic base and alkaline slant with slight H₂S production were observed, while in LIA, purple base and slant with slight H₂S production were observed (Fig. 2).

Figure 2
Result of biochemical tests. On the left, the LIA, showing purple base and slant with slight H₂S production (blackened areas). On the right, the TSI, with acidic base (yellowish), alkaline slant (reddish), and slight H₂S production (blackened areas).

The liver and spleen swab pools were also incubated at 37°C for 18 hours in Rappaport Vassiliadis broth (Biolog) and tetrathionate broth (Acumedia, Neogen Corporation, Lansing, Michigan) supplemented with novobiocin. Subsequently, the contents of the enrichment broths were streaked onto brilliant green agar (BGA) and incubated at 37°C for 18 hours. After incubation, the plates were examined, and the presence of pink colonies, non-lactose fermenters, suggestive of Salmonella spp., was noted (Fig. 3).

Figure 3
Results of streaking samples on brilliant green agar (BGA). A: Streaking from the liver and spleen culture of bird 1 in Rappaport Vassiliadis broth (RVB), after enrichment. Pink colonies indicate the multiplication of non-lactose fermenting bacteria, suggestive of Salmonella spp. B: Streaking of the liver and spleen pool of bird 2 in RVB, after enrichment. Well-defined pink colonies were observed to grow.

To confirm the suggestive result of SG from the biochemical tests, molecular tests were performed for molecular identification and differentiation of SP and SG. Duplex-PCR was conducted using the two pairs of primers previously tested by Batista et al. (2016).

Bacterial DNA extraction was carried out using the boiling method, following the methodology described by Medici et al. (2003). For this, bacteria were cultured in Falcon tubes containing 10 mL of Luria Broth (KASVI, Condá S.A. Spain), and incubated in a shaking incubator at 37°C and 150 rotations per minute (rpm) for 18 hours. After this process, 1.5 mL aliquots of each sample were transferred to 2mL microtubes and centrifuged at 13,000 rpm for 5 minutes. The supernatants were discarded, and the pellets were resuspended in 500 µL of Milli-Q Water, which were homogenized using a vortex mixer and centrifuged again under the same conditions. This process was repeated two more times, and finally, the pellets were resuspended in 100 µL of Milli-Q Water and boiled in a heating block at 100°C for 20 minutes. After boiling, the samples were subjected to an ice bath for 20 minutes. Subsequently, they were centrifuged at 5,000 rpm for 5 minutes, and the supernatant (extracted DNA) was transferred to new microtubes.

For the duplex-PCR, a solution was prepared containing the following reagents: 89.1µL of Milli-Q Water, 1X KCl buffer (15.0µL), 3.6 mM MgCl₂ (10.8µL), 240 µM deoxyribonucleotide triphosphate (dNTP) mix (18.0 µL), two pairs of primers at 0.8 µM each (2.4µL of each, totaling 9.6µL), and Long Taq DNA polymerase at 1.25 U/µL (1.5µL). The mix was equally distributed into six microtubes, adding 1 µL of template DNA into each, except for the negative control, where 1 µL of Milli-Q Water was added, totaling 25µL in each tube. A conventional thermocycler (ThermoFisher, MiniAmp - Thermal Cycler) was used with the following program: an initial denaturation cycle at 95°C for 3 minutes; followed by 26 cycles of denaturation at 94°C for 45 seconds, annealing at 63°C for 45 seconds, and extension at 72°C for 2 minutes; concluded by a final extension cycle at 72°C for 7 minutes, and cooling at 4°C for 30 minutes after the last step of the amplification cycle.

After the reaction, the amplicons were subjected to agarose gel electrophoresis, made with 1X tris-borate-EDTA (TBE) buffer and 1.5g of agarose with 10 µL of intercalating agent per 100 mL of gel. The samples were homogenized with Ludwig Dye running dye (1:5µL) and Kasvi 100bp DNA Ladder molecular weight marker. The gel was visualized under UV light, revealing two fluorescent bands in the samples originated from birds 1 and 2, consistent with the positive control for SG, suggesting a positive result for the presence of SG in both birds (Fig. 4).

Figure 4
Agarose gel electrophoresis. S1 and S2 refer to samples from birds 1 and 2, whose bands showed molecular weight similar to that of the Salmonella Gallinarum control. Molecular weight (MW), Salmonella Gallinarum (SG), Salmonella Pullorum (SP), Sample 1 (S1) and Sample 2 (S2).

After the infection was confirmed, the owner was advised to implement sanitary management measures, one of which was the emergency elimination of all birds showing clinical signs and proper dispose of the carcasses. Additionally, the entire flock was vaccinated using the 9R vaccine, with a booster dose after 4 weeks. Approximately 14 days after immunization, mortality normalized, presenting rates within the expected range according to the lineage manual.

DISCUSSION

Among the five birds sent for diagnosis, two showed apathy, prostration, and green diarrhea, which are characteristic clinical signs of FT (Freitas Neto et al., 2007; Berchieri et al., 2010). A notable and abrupt increase in mortality was observed, with rates reaching 1.8% within a single day. Despite these clinical signs and the elevated mortality, no decrease in egg production was noted, likely attributable to early detection and intervention.

Among the gross findings observed in the necropsy of the birds, changes in the ovaries, spleen, liver, heart, and lungs correlate with those described for cases of FT. Lymphoid organs atrophy and reactive cecal tonsils were also observed. Of the five birds examined, the two that showed the most evident anatomopathological changes were the ones that died during transport, indicating they were in a more advanced stage of the disease.

After the isolation and identification of SG, no antimicrobial treatment was administered. In Brazil, the treatment of fowl typhoid with antimicrobials is only permitted for laying hens and broilers (Brasil, 2003). However, even after treatment, birds may remain carriers and experience a recurrence of the disease (Barrow and Freitas Neto, 2011), demonstrating that this method is not very effective for disease control.

Associated with this, the use of antimicrobials has been widely discussed, mainly due to the risk of selection resistant bacterial strains. Won et al. (2019) analyzed the resistance of SG samples isolated in Korea to various antimicrobials and found high rates of resistance to nalidixic acid (78.5%), gentamicin (52.3%), and ciprofloxacin (26.9%). Farahani et al. (2023) evaluated the susceptibility of SG samples and found resistance to penicillin (100%), nitrofurantoin (80%), nalidixic acid (45%), cefoxitin (35%), neomycin sulfate (30%), chloramphenicol (20%), and ciprofloxacin (5%). Additionally, Sun et al. (2021) also found SG and SP isolates resistant to various antimicrobials, particularly nalidixic acid.

In this context, one of the measures adopted to control the outbreak was intramuscular vaccination with the SG9R vaccine on the fifth day after the onset of mortality, which helped the normalization of mortality and morbidity 19 days after vaccination. SG9R is a rough strain developed by Smith (1956) that has lost part of its lipopolysaccharide and, consequently, does not possess the antigenic characteristics of the smooth strain. Lee et al. (2007) demonstrated that this vaccine provided protection against mortality and organ colonization in birds challenged with SG. Although the vaccination protocol includes protection against SG, the examined birds were acquired close to the laying phase and it was not possible to ensure that they received adequate immunization at the proper age, before arriving at the farm. For this reason, a booster dose of the vaccine was recommended 4 weeks after the first dose.

Another measure implemented to reduce transmission among the birds was to remove chickens showing clinical signs of the disease and properly dispose of the carcasses. Due to its septicemic nature, the carcasses of sick birds can represent a source of contamination especially due to cannibalism (Berchieri et al., 2000). It is also important to emphasize that measures such as rodent and visitor control, water and feed quality, and cleaning and disinfection of the shed and equipment are also essential measures for preventing new outbreaks.

CONCLUSION

This case report reinforces that the removal of dead and sick hens and the administration of the SG9R vaccine are effective for the control of a fowl typhoid outbreak in laying hens, avoiding the antimicrobial use.

REFERENCES

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Publication Dates

• Publication in this collection
  28 Apr 2025
• Date of issue
  May-Jun 2025

History

• Received
  24 June 2024
• Accepted
  28 Oct 2024