The liver is considered, together with the intestine, a key organ in intensive poultry farming, since it is responsible for two fundamental functions such as detoxification, and the synthesis of proteins, carbohydrates, and lipids [1].

The first line of defense is mainly exercised by the mucous membranes, which protect the animal from several external antigens, and in this sense, the intestinal and the respiratory ones, as well as the skin are the most relevant. However, several antigens, including many microorganisms, manage to cross these mucous membranes, reach the bloodstream, and affect the liver, a key organ due to its synthesis function, but it is important to note in this case, that it acts as a second line of defense against these pathogens [2].

Multiple bacteria have been isolated from birds’ liver along the past, and in recent decades, the incidence of bacterial liver diseases has increased due to many causes. Genetic lines are under high selective pressure to achieve great productive performance. This entails an increase in metabolic demand, with a higher feed intake and, therefore, a very important effort of the intestine, which is in contact with multiple antigens that end up exceeding its defensive capacity and reaching the liver. On the other hand, high densities can cause certain microorganisms to penetrate through the skin or the respiratory mucosa, eventually reaching this organ. In the case of breeders, additionally, they can spread these bacteria through vertical infection to subsequent generations, which aggravates the problem [3].

It is also necessary to take into consideration that, although many of these bacteria are well known and have been isolated for years, antibiotic programs have lost efficacy due to the increase in antimicrobial resistance, and there are also errors in clinical practice when addressing liver infections: once the bacteria settle in the liver, and after having performed a corresponding antibiogram after their isolation, the antibiotic of choice must complete the enterohepatic cycle to be effective, otherwise it will not reach the bacteria and will contribute to the development of more microbial resistance.

Finally, it is necessary to highlight that, although from a clinical point of view at the farm level, the most interesting bacteria are those pathogenic for the birds, such as Salmonella pullorum, S. gallinarum, Clostridium perfringens, etc. there are many others that are found in the liver with zoonotic capacity, and some emerging ones that must be taken into consideration, such as many serovars of Salmonella or C. hepaticus [4].

A bet of the VallBo Foundation for the investigation of bacteria with liver multiplication in conditions of Thailand

Up to now, in poultry, there were only brief prevalence reports of certain liver bacteria in Thailand, where a high prevalence of Salmonella (84%) was reported, but in this case from raw meat [5]. In other countries, such as Argentina [6] or the United States [7], certain prevalence levels were determined in the liver.

Since until now there was no evidence of bacterial isolation from chicken liver samples in Thailand, VallBo foundation decided to bet on this research, highly important for one of the sectors with highest impact in the country. The research was conducted by researchers from Chulalongkorn University. The verification of bacterial diversity in liver samples in farms and slaughterhouses is necessary to identify the most common bacterial pathogens in poultry production. The results of this study will help to identify problems on farms, as well as those of zoonotic importance. Therefore, the objectives of this study were to identify those bacteria, both pathogenic for birds and zoonotic, including E. coli, Salmonella spp., Shigella spp., Campylobacter spp., Staphylococcus spp., Streptococcus spp., P. multocida and C. perfringens, isolated from liver, and sampling from farms and slaughterhouses, during the year 2020.

Experimental design of the and results

Three hundred ten (n=310) samples of livers were collected from farms (n=160) and slaughterhouses (n=150) from the different regions of Thailand, selected with an adequate sampling. Bacteria isolated from chicken liver samples were classified by their origin, that is, those from farms or slaughterhouses, as well as the mean prevalence of each target bacteria in these locations, both separately and in total.

The most commonly isolated bacteria in chicken liver samples, in general terms, were E. coli (80.65%), followed by Streptococcus (45.48%), Salmonella (15.81%), and Campylobacter (14.52%), respectively. A low prevalence (<10%) of Shigella (5.81%) and Staphylococcus (4.52%) was observed. On the other hand, in this study neither P. multocida nor C. perfringens were detected (although it is true that the latter is more frequent to observe the toxins reaching the liver rather than the bacteria itself).

If we analyze the results by differentiating the prevalence between farm and slaughterhouse isolates, we can see that, on farms, the most common pathogens were E. coli (72.50%), Streptococcus (12.50%), Salmonella (8.13%) and Campylobacter (8.13%). On the other hand, the predominant microorganisms isolated from slaughterhouse samples were E. coli (89.33%), Streptococcus (80.67%) and Salmonella (24.0%). The distribution of pathogens identified between farms and slaughterhouses was statistically significant for Streptococcus spp (P <0.0001), Escherichia coli (P <0.0001), Salmonella (P=0.0001), Shigella (P=0.002) and Campylobacter (P=0.001)). On the other hand, there was no significant difference in the distribution of Staphylococcus between farms and slaughterhouses. In general terms, from this significance it can be established that greater contamination is found in livers from slaughterhouses for all the bacterial species analyzed. This is perhaps since, in processing plants, and especially in the evisceration process, bacteria from other locations of the bird can contaminate the liver, especially those coming from the intestine, that can be broken by the mechanized process of extraction of the gut.

It is necessary to highlight that, as can be seen in the results (table 1), when the sum of the percentages of the isolates is obtained, it is observed that 166.8 microorganisms are isolated from every 100 samples (in absolute terms, 517 isolated in the total out of 310 samples). This means that 66.8% of the samples present more than one contamination and it is essential to perform pathogenicity tests to the bacteria, in order to differenciate pathogens from those saprophytes that do not cause disease. For this there are two types of tests:

• Biochemical tests: such as SH2 +, mannitol +, methylene blue reduction, or hemolysis on blood agar.
• In vivo tests: intraperitoneal injection of an 8-hour culture in BHI (brain-heart infusion) for Gram + bacteria or 6 hours for Gram – bacteria.

These tests are of great relevance, since isolating a microorganism is not enough to consider it a cause of disease, a confirmation must be performed by using some of the techniques described.

Other results of interest derived from the research

Other interesting conclusions were drawn from the study, especially regarding multiple infections, the characteristics of the affected livers and the typification of the isolated Salmonella strains.

It was observed that, in 66.8% of the samples multiple microorganisms were isolated simultaneously. However, some samples multi-contaminated with the same bacterial species may present a very different appearance. This can be due to multiple factors, such as the nutritional and health status of the bird, or the bacterial load.

At a macroscopic level, it should be noted that, among the livers that showed macroscopic alterations, those congestive were the most prevalent, and some of them also presented altered consistency, were friable, and had petechiae or clots.

However, as can be seen in Table 3, most of the livers presented a normal appearance. This may be due to non-pathogenic bacteria for the bird itself, which may be zoonotic, or not advanced stages of infection, when the pathology has not yet developed in the bird. It can also be associated with bacteria that reach the liver, not being the target organ where they cause their pathogenic effect.

In addition to bacterial isolation, in the case of Salmonella, serotyping was carried out to determine the prevalence of the different serovars. For this, a total of 34 liver samples were collected and a total of 21 different serotypes of Salmonella were obtained (Table 5).

Among the 21 Salmonella serotypes isolated from broiler liver samples, the predominant serotypes were Schwarzengrund (17%), followed by Agona (15%) and Stanley (13%). These Salmonella serotypes are of zoonotic importance, of avian origin, and, therefore, knowing this prevalence data can be of great help when implementing prevention and control measures.

Conclusions

The liver is a key organ in intensive poultry farming, which is in contact with many antigens, due to its detoxifying function, and many microorganisms manage to reach it after passing through some mucous membranes of the animal. Many types of bacteria have been isolated over the past decades from liver, and in recent decades, the incidence of bacterial liver diseases has increased considerably.

It is also necessary to highlight that, although from a clinical point of view, the most important bacteria are those pathogenic for the birds, there are many others that are found in the liver with zoonotic capacity, such as many Salmonella serovars.

VallBo Foundation, last 2020 supported a research of the main bacteria isolated in broiler livers in conditions of Thailand, carried out at the University of Chulalongkorn. In the results, it was observed that the main isolated bacteria were, in general, E. coli (80.65%), followed by Streptococcus (45.48%), Salmonella (15.81%), and Campylobacter (14.52%), respectively. In addition, 21 Salmonella serotypes with zoonotic importance were found, and it was determined that these bacteria were isolated from livers with a normal macroscopic appearance in more than 50% of the cases, but many others presented alterations, such as congestion, paleness or showed clots and gas. This indicates that the isolated bacteria are of both types, both zoonotic and pathogens for the birds.

These findings will allow the knowledge of a current and precise scenario of the microorganisms that affect birds, as well as those that can affect humans through the food chain. This will allow the implementation of prevention and control measures that improve health in broiler and breeder farms, and enhance food safety by controlling those pathogenic bacteria.

Bibliography

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[2] Borrell, J. Tesouro A. Díez, D. “Digestive ecology, dysbiosis and pronutrients”. 2021. Academia Peruana de Ciencias Veterinarias

[3] Díez, D. “Integrity of the intestinal epithelium”. 2020 www.veterinaria digital.com

[4]  Courticea JM, Mahdia LK, Grovesb PJ, Kotiwa M 2018. Spotty Liver Disease: A review of an ongoing challenge in commercial free-range egg production. 227: 112-118.

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