Detection of Erysipelothrix rhusiopathiae in naturally infected pigs in Kamuli District, Uganda

Swine erysipelas, or ‘diamond skin disease’, is an economically significant disease affecting all stages of pig production. The biggest losses may occur in growers-finishers because of sudden death or acute septicemia. Survivors often suffer from chronic lameness, arthritis and endocarditis leading to poor body growth or infertility. The causative agent is the ubiquitous bacterium Erysipelothrix (E.) rhusiopathiae. It is distributed worldwide and primarily considered as an animal pathogen. Turkeys and pigs are most commonly affected, followed by other birds, sheep, fish and reptiles. E. rhusiopathiae is zoonotic, and transmitted through scratches or small injuries of the skin. It is not commonly known among researchers, veterinarians, personnel of the health care sector in general, butchers, abattoir workers, farmers, fishermen, fish handlers and housewives. It has been referred to as an occupational disease by Wood (1975), as it affects these categories of people who work closely with infected animals or their products. In humans, it usually manifests itself as localized cellulitis known as erysipeloid. If infections get systemic, patients may have fever, joint pain and lymphadenopathy. Infections in otherwise healthy people are usually self-limited within three to four weeks (Reboli and Farrar, 1989). In rare cases, E. rhusiopathiae causes septicemia and potentially fatal endocarditis.


■ INTRODUCTION
Swine erysipelas, or 'diamond skin disease', is an economically significant disease affecting all stages of pig production.The biggest losses may occur in growers-finishers because of sudden death or acute septicemia.Survivors often suffer from chronic lameness, arthritis and endocarditis leading to poor body growth or infertility.The causative agent is the ubiquitous bacterium Erysipelothrix (E.) rhusiopathiae.
It is distributed worldwide and primarily considered as an animal pathogen.Turkeys and pigs are most commonly affected, followed by other birds, sheep, fish and reptiles.E. rhusiopathiae is zoonotic, and transmitted through scratches or small injuries of the skin.It is not commonly known among researchers, veterinarians, personnel of the health care sector in general, butchers, abattoir workers, farmers, fishermen, fish handlers and housewives.It has been referred to as an occupational disease by Wood (1975), as it affects these categories of people who work closely with infected animals or their products.In humans, it usually manifests itself as localized cellulitis known as erysipeloid.If infections get systemic, patients may have fever, joint pain and lymphadenopathy.Infections in otherwise healthy people are usually self-limited within three to four weeks (Reboli and Farrar, 1989).In rare cases, E. rhusiopathiae causes septicemia and potentially fatal endocarditis.
In Uganda, pig keeping has emerged as a popular income-generating activity over the past three decades (MAAIF/UBOS, 2009).It is the first East African country in terms of consumption per capita (FAOSTAT, 2011).Nevertheless, many of the potentially zoonotic pig diseases have never been researched in Uganda (Alonso et al., 2016) because generally pigs have played a negligible role in agricultural production in the past in the country (Blench, 2000).
Preliminary information from participatory rural appraisals conducted in 2013 indicated the occurrence of diamond skin disease in pigs in Kamuli District, Eastern Uganda (Roesel et al., 2014).According to these appraisals, farmers from three subcounties had reported skin lesions in live pigs, which may have been caused by E. rhusiopathiae; the disease is locally referred to as 'Okumyuka'.In the present study, we investigated the existence of E. rhusiopathiae in pigs and pork in the villages that reported signs of the disease.

Study area
The study was conducted in Kamuli District, Eastern Uganda, and was part of a multidisciplinary program implemented by the International Livestock Research Institute (ILRI) and partners to identify constraints and opportunities to improve smallholder pig value chains in Uganda (Ouma et al., 2014).The district has an estimated population of 55,998 pigs and most pigs are kept under extensive or semiextensive systems with free-ranging and tethering being the most practiced keeping types.

Sample collection
Serum aliquots from 426 randomly selected pigs in four subcounties, namely Bugulumbya, Butansi, Kitayunjwa, Namwendwa, in Kamuli District (Figure 1), had been obtained in previous investigations (Erume et al., 2016) that were part of a multipathogen survey led by ILRI.Subsequently, 100 samples of fresh pork were collected from all 67 slaughterhouses operating in the same subcounties at the time of the study in March 2014.Using a sharp knife and gloves, approximately 250 grams of small pieces of pork from deep inside the muscle were cut.After collection, the pork was put in a stomacher bag, labelled and sealed with tape.It was put on ice in a cool box and transported to the College of Veterinary Medicine, Animal Resources and Biosecurity for microbiological analysis, which was carried out within ten days.

Laboratory analysis Indirect enzyme-linked immunosorbent assay
A commercially available ELISA (CIVtest Suis SE/MR, Laboratorios Hipra, Amer, Spain) based on E. rhusiopathiae-specific antigen (E.rhusiopathiae R32E11, serotype B) was used to detect anti-Erysipelothrix spp.immunoglobulin G.According to the manufacturer's instructions, the assay showed a sensitivity and specificity of 100%.The 96-well microplates were provided precoated with antigen, and the assay was performed as follows: 50 µl of the undiluted, readyto-use controls and 1:200 diluted test samples were added to the appropriate wells and left to incubate for 60 min at 37°C to allow antigen-antibody binding.All wells were washed three times with reconstituted washing solution (300 µl) and, subsequently, 50 µl of a ready-to-use conjugate solution (MAb anti-porcine IgGs/HPRO) were added to each well and left to incubate for another 60 min at 37°C to bind any attached porcine antibody.Remaining unbound conjugate was washed three times (300 µl) in another step, before a peroxidase-specific chromogenic substrate solution (50 µl) was added to each well and left in the dark at room temperature for 15 min to allow a chromogenic reaction to develop.The reaction was stopped by adding 50-µl ready-to-use oxalic acid solution.
Optical density (OD) of the sample was measured by reading the test plate at 405 nm wavelength (ELx800, BioTEK; Gen5 2.00.18).For the interpretation of results, a relative index (IRPC) value was required (IRPC = [(sample OD -mean negative control OD / mean positive control OD -mean negative control OD)] * 100, where an IRPC value above 40.0 was defined as positive, according to the manufacturer.All samples were analyzed in duplicates using the positive and negative controls provided by the manufacturer.

Culture isolation and identification from pork samples
Bacteriological culture was performed according to standard procedures (Reboli and Farrar, 1989).Briefly, pork samples were homogenized in a sterile stomacher bag.Five microliters of the resulting solution were streaked on sterile trypticase soy agar (laboratorios-Conda, Madrid, Spain) and modified blood agar containing 0.4% sodium azide (Laboratorios-Conda) and incubated for 24-48 hours at 37°C.The plates were examined after incubation for suspect E. rhusiopathiae colonies which were pinpoint size (approximately 0.1 mm), convex, circular, translucent, smooth, mildly hemolytic colonies (Reboli and Farrar, 1989).The colonies with morphological characteristics of E. rhusiopathiae were subcultured on Erysipelothrix selective media (Laboratorios-Conda) as reviewed by Wang et al. (2010) and subsequently incubated for 24 hours at 37°C.Bacterial colonies that grew on the selective media were identified by their morphology: small (0.3-0.6 µm), circular, and transparent, with a smooth glistening surface and edge, and by Gram staining.Subsequently, they were biochemically confirmed by testing for catalase activity as well as their ability to hydrolyze esculin and break down protein gelatine (Forbes et al., 2007).

Data management and analysis
Laboratory data and data collected on demographics (location of the butcher, location of the pigs) were double entered in Excel and crosschecked for completeness and accuracy before descriptive analysis in STATA 12.0 (StataCorp).Confidence limits were computed with Ausvet EpiTools (http://epitools.ausvet.com.au/content.php?page=CIProportion) for a desired level of confidence of 95% using the binomial (Clopper-Pearson) 'exact' method based on beta distribution.Significance in prevalence between villages was assessed based on the overlap of confidence intervals (Tables I and II).

■ RESULTS
In the first step, ELISA revealed an overall seroprevalence of antibodies against E. rhusiopathiae in pig sera of 66.9%, with no significant differences between subcounties (Table I).
In the second step, results showed that E. rhusiopathiae was isolated from 45% of the 100 raw pork samples with the highest proportion found in Butansi subcounty (Table II).However, differences between subcounties were not statistically significant.

■ DISCUSSION
To the knowledge of the authors, this is the first report of the occurrence of E. rhusiopathiae in pigs and pork in Uganda, and the bacterium has been isolated from all subcounties in the study where farmers had described clinical signs of acute swine erysipelas during systematically conducted participatory research (Roesel et al., 2014) therefore offering potential for increased income through smallscale pig production and marketing.A multi-disciplinary value chain assessment conducted by ILRI and partners aimed to identify constraints and opportunities for value chain actors as well as shortcomings in the safety of pork products in three districts in Uganda.Prior to quantitative surveys and biological sampling at various nodes of the chain, participatory rural appraisals and focus group discussions were held with about 1,400 smallholder pig farmers to map out qualitative aspects including the various actors involved in pig rearing (e.g.input and service providers).In the East African Community, E. rhusiopathiae has been previously isolated from pigs in Kenya (Wabacha et al., 1998;Friendship and Bilkei, 2007), where it caused high mortality suggesting a virulent serovar affecting pigs in the region.Pigs have been shown to harbor both avirulent and virulent serotypes in their tonsils (Takahashi et al., 1987).Although clinical cases of swine erysipelas are predominantly caused by serotypes 1a, 1b, or 2, less common serotypes typically have lower virulence for swine (Opriessnig and Wood, 2012).The current aim of the study was to reveal the presence of E. rhusiopathiae in pigs but not to serotype the isolates.However, given the unexpectedly high prevalence in raw pork, the serotypes circulating in Kamuli District should be determined in future research.
Infection with E. rhusiopathiae in pigs can be present under three known forms of the disease, depending on the virulence of the serotype and the host's immunity status: acute, subacute and chronic.The acute form is characterized by a septicemia with sudden fever, abortion, lethargy, lying down, stiffness of joints, partial or complete lack of appetite, characteristic pink, red or purple, raised, rhomboid or squared skin lesions around the snout, ears, jowls, throat, abdomen and thighs, and death (Opriessnig and Wood, 2012).Especially in dark-skinned pigs, where the characteristic shape of the skin lesion may not be fully visible, many of the symptoms may be confused with signs of African swine fever which is perceived to be a major health constraint to smallholder pig production in the region (Dione et al., 2014).On the other hand, the subacute form of the disease may result in infertility in pigs, whereas the chronic form may cause lameness; both conditions can cause severe production losses.However, there are both treatment and immunization available for the control of to identify control strategies, especially vaccination, that could be implemented in Kamuli District.It should also attempt to assess the burden of E. rhusiopathiae infection in raw pork handlers such as butchers and cooks, and identify practices that might increase the risk of human infection.Public health practitioners in Eastern Uganda such as physicians, nurses, veterinarians and meat inspectors should be sensitized on the presence of the pathogen as well as its diagnosis and control.Much remains to be elucidated on the epidemiology and transmission cycle of E. rhusiopathiae in animals, people and the environment in Uganda.Future investigations should focus on the One Health approach.and butchers in Kamuli District for their participation in the survey.
E. rhusiopathiae infection in pigs, but the latter requires knowledge on the circulating serotypes.In humans as in pigs, the disease can easily be treated but only if detected in time.

■ CONCLUSION
As clinical signs of swine erysipelas had been reported by smallholder pig farmers in Kamuli District, the study aimed at showing the prevalence of the disease in the area.We showed that a 66.7% seroprevalence was found among the 426 pigs investigated, and a 45% prevalence was found in pork samples from the 67 slaughterhouses.
Therefore, E. rhusiopathiae is present in pigs in Kamuli District, posing an economic risk to smallholder pig farmers, and a potentially public health risk to pig and pork handlers in the area.Moreover, we were able to confirm that community-based participatory disease reporting is helpful in identifying diseases in livestock species that are historically recent in developing countries.

Figure 1 :
Figure 1: Selected subcounties for detection of Erysipelothrix rhusiopathiae in pigs and pork in Uganda.