Journal of Veterinary Science & Medical Diagnosis ISSN: 2325-9590

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Research Article, J Vet Sci Med Diagn Vol: 5 Issue: 2

Rejections due to Bacterial Infections in an Ovine Abattoir

Vilallonga D and Valcárcel F*
Parasitology Group, Animal Health Research Center (INIA-CISA), 28130-Valdeolmos, Madrid, Spain
Corresponding author : Félix Valcárcel
Parasitology Group, Animal Health Research Center (INIA-CISA), 28130-Valdeolmos, Madrid, Spain
Tel: 0034 91 620 23 00; Fax: 0034 91 620 22 47
E-mail: [email protected]; [email protected]
Received: November 07, 2015 Accepted: March 03, 2016 Published: March 10, 2016
Citation: Vilallonga D, Valcárcel F (2016) Rejections due to Bacterial Infections in an Ovine Abattoir. J Vet Sci Med Diagn 5:2. doi:10.4172/2325-9590.1000191

Abstract

Rejections due to Bacterial Infections in an Ovine Abattoir

Over the course of a year, organs rejected during meat inspection were sampled from an ovine abattoir that slaughtered both sheep and lambs. The aim of this study was to determine which bacterial diseases go unnoticed throughout a sheep’s useful, and to demonstrate how basic laboratory equipment can be used to develop bacteriologic protocols that detect common bacterial species, ultimately helping the abattoir management system prevent disease from reaching the human food chain. Rejections from meat inspection were processed in a veterinary laboratory to determine the final diagnosis and the etiology of such rejections. The ovine pathologies found were few in number and very dependent on the age of the animal. The bacterial disease that was most prominent for its high prevalence was enzootic pneumonia, followed by liver and lung abscesses, caseous lymphadenitis, and other lung processes like purulent lymphadenitis and pneumonitis. Season was found to be an important variable, particularly in cases of enzootic pneumonia and caseous lymphadenitis. However, mycoplasma infections needed more specific and complex techniques in order to be detected.

Keywords: Slaughter; Bacterial infections; Ovines; Public Health

Keywords

Slaughter; Bacterial infections; Ovines; Public Health

Introduction

Rejections during meat inspections in slaughterhouses result in great economic disturbance to farmers. In most cases, these rejections are caused by a wide range of processes: abscesses, wasting or cachexia, ante mortem demise, caseous lymphadenitis, helminthosis, hydatidosis, sarcosporidiosis, jaundice and pneumonia in adult sheep, while in lambs the main causes are wasting, arthritis, ante mortem demise, feverish meat, abscesses, cysticercosis and jaundice [1-3]. However, there is a limited group of pathologies that stand out due to their higher frequency of presentation [4] as this study shows.
Although these common bacterial processes are usually multifactorial, there are one or two primary agents involved in the process. The main bacterial agents involved in the ovine enzootic pneumonia, excluding micoplasms, are several strains of Mannheimia haemolytica , although from the lesions, occasionally, other organisms can be isolated: Pasteurella multocida , Bordetella parapertussis and Branhamella catharralis [5-10]. Mycoplasma spp. has been isolated form ovine pneumonic lungs by several researchers [11-13]. Although its presence is not always accompanied by pulmonary damage, some authors consider these agents the cause of mild initial and low grade lesions in the pulmonary tissue that facilitate the later action of other microorganisms like M. haemolytica and P. multocida [14,15]. Fusobacterium necrophorum is the most important agent involved in localized purulent processes in liver and lungs [16] along with Dichelobacter spp . [17]. However, other bacteria such as Arcanobacterium pyogenes , Escherichia coli , Enterococcus spp , Enterobacter spp , Pseudomonas aeruginosa , Staphylococcus aureus , Streptococcus spp ., etc. are easier isolated [18]. The corinebacteria of interest in meat inspection include A. pyogenes, agents of purulent infectious processes, and Corynebacterium pseudotuberculosis , which produces pseudotuberculosis or ovine caseous lymphadenitis [19]. The differential diagnostic with other pathologies, which show up with abscesses of similar features, is of high importance. Among them, infections caused by S. aureus , Streptococcus spp . and A. pyogenes [20-22] should be considered. The microbiological studies of the disease of abscesses, also known as enfermedad de Morel, have determined that the etiological agent that causes the condition is S. aureus anaerobius [23,24].
In this context, there is a clear interest in establishing the underlying bacterial etiology of the rejections. However, due to the dynamics of working in slaughterhouses, it is usually difficult to uncover this etiology. Furthermore, there are mismatches in the lists of reasons or causes leading to rejections and in the percentages attributed to each reason. The aim of this study was to create an updated approach to the etiology of the main bacterial diseases that account for the vast majority of rejections in an ovine slaughterhouse and to develop simple bacterial protocols that identify common bacterial agents involved using basic laboratory equipment.

Materials and Methods

Place of study and sample recovering
The study was conducted between October 2010 and September 2011 at a slaughterhouse in Madrid, in the central area of Spain. A monthly batch of lambs and adult sheep older than two years of age (40-50 and 10-20 heads) was selected. The rejections were initially identified and classified according to the preliminary identification discussed in the next section and pictures were taken. Immediately following rejection by the meat inspection, a sample of confiscated tissue was obtained at the slaughter line with a sterile scalpel to avoid cross contamination, then placed in a sterile, transparent plastic bag and finally stored at -20°C.
The samples were then transported to the laboratory in a cooler to maintain temperature. Once in the laboratory, the samples were allowed to thaw at room temperature (20-24°C) in their individually packed, sterile plastic bags and were again subjected to a macroscopic examination.
Preliminary identification of rejections
Rejected samples were photographed and grouped into appropriate pathology categories:
Lesions compatible with enzootic pneumonia (confirmed later by bacteriological identification of M. haemolytica , P. multocida , B. catarrhalis and B. parapertussis );
Lesions compatible with pulmonary caseous lymphadenitis and pulmonary abscesses (confirmed by bacterial identification of C. pseudotuberculosis , A. pyogenes , P. aeruginosa , Streptococcus spp. , S. aureus aureus and S aureus anaerobius );
lesions compatible with hepatic abscesses, caseous lymphadenitis, hepatitis and hepatic necrosis (confirmed later by bacterial identification of Fusobacterium spp ., Dichelobacter spp ., C. pseudotuberculosis , A. pyogenes , P. aeruginosa , Streptococcus spp ., S. aureus aureus and S. aureus anaerobius ).
White focus that was enclosed by a capsule with a centre of a white or greenish hue and produced malodorous purulent material when cut, including caseous formations with fibrous onion-like capsular layers were noted purulent or calcified abscesses. When it was observed the growth of confirmed C. pseudotuberculosis these lesions were noted as caseous lymphadenitis.
"Other liver lesions "or "other lung lesions" were noted when these bacterial species were not identified.
If S. aureus anaerobius growth was observed, it was considered cases of purulent abcess disease.
Bacterial identification
Bacterila identification entailed bacterial culture, bacterial staining from the lesion and the resulting culture, and biochemical testing. Gram and carbol basic fuchsin stains, a catalase test, and oxidase techniques were performed following the manufacturer's instructions and the recommendations of Quinn et al. [25]. Bacterial cultures were performed in sheep blood in aerobiosis and anaerobiosis and McConkey agar. Samples with no bacterial growth were classified as "no growth". To determine the presence of micoplasma species, 60 samples were processed by a PCR-ELISA from Roche Laboratories. The technique was performed following the laboratory instructions.
Data analysis
The data were processed using the SPSS 17.0 statistical software for Windows (SPSS Inc., Chicago, IL USA) and Epi-Info 4.0. To test the associations between variables, a Z proportions comparison test was used. Moreover, the OR and confidence intervals were determined at 95%.

Results

A total of 1,945 lambs and 484 adults were analyzed, representing 3.82% and 1.70% of the sacrifice volume in the slaughterhouse (0.02% and 0.07% of the animals slaughtered nationwide).
347 (60.14%) of 577 rejected organs (lungs, liver, lymph nodes and muscles) had a bacterial condition as the cause of the condemnation totalled. The remainder had either a parasitic origin or some other cause (38.13% and 1.73%) [26,27].
In this study, 11% of the lambs and 29% of the adults inspected had a bacterial condition as the primary reason for condemnation, all of which were related to liver and lung diseases.
The bacterial diseases that we could identify were enzootic pneumonia in both adults and lambs, followed by liver and lung abscesses, with a higher prevalence in adults than in lambs. In adults, caseous lymphadenitis was also identified, as were purulent lymphadenitis and pneumonia, although the last two were observed at a much lower frequency.
The highest frequency of condemned adults and lambs was found in spring and summer (approximately 15% and 39% of lambs and adults, respectively), whereas winter was the season with the smallest proportion of bacterial rejections (Tables 1 and 2).
Table 1: Seasonal distribution of rejections caused by bacterial infection in lambs in an abattoir in central Spain during October 2010 to September 2011.
Table 2: Seasonal distribution of rejections caused by bacterial infection in adult sheep in an abattoir in central Spain during October 2010 to September 2011.
The bacteria that were isolated and identified in samples taken from rejections by all others pathologies are detailed in Tables 3 and 4.
Table 3: Bacterial growth in cultures from lamb rejections sampled in an abattoir in central Spain during October 2010 to September 2011.
Table 4: Bacterial growth in cultures from adult sheep rejections sampled in an abattoir in central Spain during October 2010 to September 2011.
The mean annual percentage of rejections due to enzootic pneumonia was 7.87% in lambs and 9.30% in adults. Spring and summer were the seasons of greatest presentation in lambs (χ2=21.5345; p=0.0001), and summer and autumn were the seasons of greatest presentation in adults (χ2=12.6647; p=0.0054). In both lambs and adults, the most infrequent season of presentation was the winter. In lambs, the percentage of rejections was significantly lower in winter and autumn than in spring and summer (χ2=21.5345; p=0.0001). In adults, the season also had a significant influence (χ2=12.6647; p=0.0054), with the highest percentage occurring in the summer and the lowest occurring in the winter. We only observed a concomitant pathology of enzootic pneumonia rejections with purulent abscesses in adults
The main pathogen isolated from lamb pneumonic tissue was M. haemolytica , which was present in 78.10% of the cultures, followed by P. multocida and B. catarrhalis and, at much lower levels, B. parapertussis and A. pyogenes.
The annual incidence of liver abscesses was 2.47% in lambs and 6.61% in adults. The largest percentage of rejections occurred in summer, followed by autumn in both lambs (χ2=2.5893; p=0.4594) and adults (χ2=3.5954; p=0.3086). There were no statistically significant differences between seasons, though. Other lung lesions were observed in almost 7% of adults and less than 1% of the lambs, with spring being the season with the most rejections by this cause in lambs (χ2=2.9624; p=0.3975), although with no statistical significance, and in sheep (χ2=14.9983; p=0.0018).
The concurrent pathologies and injuries found to be associated with each case of lung abscess in adult sheep were hydatidosis and enzootic pneumonia. S. aureus was isolated in over half of lung abcess cultures, followed distantly by Fusobacterium spp ., Streptococcus spp . and other unidentified pathogens. This pattern was similar in sheep and lambs, but in the case of adults, the unidentified microorganisms had a higher percentage of prevalence (17.95% and 34.78%, respectively).
Approximately 5% of adult lungs were condemned due to caseous lymphadenitis, with the highest incidence occurring in spring (11.90%) and the lowest in autumn (1.11%), which was significantly different (χ2=16.0244; p=0.0011). The most frequent form of the disease was the presence of caseous foci in bronchial and mediastinal lymph nodes. Lymph nodes, which acquired a considerable size, had thickened capsules and purulent content, with varied consistency. This process was not detected in lambs. Among the microorganisms isolated from the lesions caused by caseous lymphadenitis, C. pseudotuberculosis was prominent and found in 100% of lesions consistent with caseous lymphadenitis, with occasional contamination by S. aureus or other germs. In spring, we found a case of the disease of abscesses concomitant to caseous lymphadenitis. The culture of this condemnation tested positive for S. aureus anaerobius and C. pseudotuberculosis growth.
The percentage of Mycoplasma spp. isolations has been very consistent throughout the year, with a small peak in the summer (54.55%) and an annual mean of 44.68% (Table 5).
Table 5: Isolation of Mycoplasma spp. in ovines.
Other important pathologies detected were hydatidosis in sheep and cysticercosis in lambs [28,29], with rejections associated with a higher variety of pathologies and lesions, in both cases, highlighting the abscesses that were either purulent or calcified. S. aureus was isolated in over half of hydatid cysts and 70% of the lesions caused by cysticercosis, followed by other pyogenic bacteria such as Streptococcus spp ., A. pyogenes and C. pseudotuberculosis.

Discussion

The incidence of rejection was higher in adults than in lambs, which makes sense given that the former have been exposed to increased number of infections by various pathogens. These percentages appeared to be relatively high, considering that many sick animals are detected while in their own herds and does not reach the slaughterhouse [30-33].
The fact that all detected bacterial processes were related to liver and lungs in both lambs and adults is in agreement with several studies noting that these organs are the most frequent causes of condemnation in ovine slaughterhouses. Similarly, despite the variety of reasons for condemnation, only a few bacterial diseases or processes—enzootic pneumonia and abscesses in this study—are found in most rejections, as has been previously described [34].
Enzootic pneumonia
The annual prevalence of enzootic pneumonia in lambs was similar to that reported previously [35-37]. The estival tendency towards rejections by this cause in adults was in concurrence with the results of Arrigo et al. [38] and Martin and Aitken [39], who reported that most outbreaks of pneumonic pasteurellosis in Europe presented from May to July and that many of these outbreaks affected both adult sheep and lambs. Interestingly, as Dungworth [40] noted, the seasonality of the disease coincides with the presence of microscopic lesions consistent with severe bacterial contamination (94.10% of lesions in lungs in summer, data not shown).
Other authors have also found a direct relationship between pneumonia in lambs and the warmer seasons, spring and summer, which are times when there is an increased mortality and emergency slaughter in farms [41]. In contrast to these findings, some studies have found high prevalence in autumn-winter [42], whereas others have found no significant variation between rainy and dry seasons [43]. Because members of the Pasteurellaceae family can occur at any time of year, it appears that a wide range of intermediate scenarios may be observed.
Other studies have suggested that there is no real seasonality in the presentation of enzootic pneumonia because it is possible to associate the disease with the abrupt climate changes that occur in early summer or winter [44] and with sudden changes in intraday temperature [45,46] or the presence of rainfall, wind speed, temperature and humidity, particularly the rain/wind chill factor, rather than with certain seasons. However, other factors that influence the manifestation of the disease should be considered, including the infectious agents themselves and the host [47], overcrowding [48,49], the mixture of animals on farms of different ages and immunological levels, excessive heat or cold, high humidity, long journeys, poorly ventilated facilities, presence of high concentrations of pollutants in the air, castration, sudden changes in supply [50,51].
The percentage of Mycoplasma isolation is very similar to the ones recorded by other Spanish authors like Gracia et al. [52], who found percentages of isolation of 40.00% for M. ovipneumoniae and 33.00% for M. arginini. Other national authors show smaller figures of 24.00% for M. ovipneumoniae and 4.70% for M. arginini. The results of this study support the hypothesis advanced by several authors that certain species of the genus Mycoplasma spp. can cause an initial infection and mild pneumonia and respiratory infection, leading to an increase in the sensitivity of ovines and caprines to secondary infections by M. haemolytica and, less frequently, P. multocida or other minor microorganisms [53-55]. The coincident purulent abscess with enzootic pneumonia is most likely an evolution of the initial pneumonia; thus, after the infection becomes chronic, the local immune response could encapsulate the lesions with pyogenic organisms that ultimately develop into abscesses.
The identification of bacteria by lamb pneumonic tissue culture appeared to be variable. Our findings of M. haemolytica followed by P. multocida and B. catarrhalis and, at much lower levels, B. parapertussis and A. pyogenes are similar to those found by other authors [56]. Other studies, however, have reported that the isolation of M. haemolytica and P. multocida is less important when culturing pneumonic tissue in lambs [57]. Interestingly, although there is generally less isolation in adults than in lambs, isolates of M. haemolytica remain the most frequent in lesions caused by enzootic pneumonia, followed by P. multocida and B. catarrhalis. Differences between countries could exist because in the previously mentioned studies, the lambs that were sent to slaughter were significantly older (6 months to 1 year of age) than in this study (less than 4 months old). It is noteworthy that the percentage of isolation of M. haemolytica from pneumonic lungs of adults in Spain is very similar to that of lambs abroad and Sisay and Zerihun [58] also found high percentages in Turkey and Ethiopia, respectively.
Abscesses in liver
The annual prevalence of liver abscesses in adults was twice that of lambs, confirming that the age of lambs at slaughter may be an important factor to consider. This is coincident with the results of Edwards et al. [56] who indicated that the age and environmental factors more heavily influenced the presence of lesions than did health and disease variables. The occurrence of abscesses may also be related to other factors such as postpartum hygiene in sheep or the health status of the farm of origin.
The high presence of S. aureus , Streptococcus spp ., A. pyogenes and C. pseudotuberculosis in cultured hydatid cysts and S. aureus in cysticercosis samples suggests a possible association between the presence of metacestodosis and liver abscesses. Thus, because an undetermined percentage of abscesses (liver or lung) may be secondary to hydatid cysts, it is possible that the prevalence of this condition is consistently underestimated. The organic migration performed by tapeworm larvae may host pyogenic bacteria, or these lesions may become calcified after a chronic inflammatory process before evolving into an abscess. Even the parasite migration itself may turn into these purulent or calcified tracts in the case of cysticercosis. The tracts that this parasite produces are haemorrhagic at the onset, and the purulent tracts are an evolution of those that are followed by an S. aureus infection.
Thus, the question arises on which route has been followed by these isolated pathogens to reach those hepatic locations. Such bacteria may have been transported by larval forms of tapeworms from outside the animal, through their digestive tract to its final location in the liver, or may simply carry one or more germs that were already dormant in the host, favouring the damage caused by the metacestode proliferation and/or dissemination.
The other isolates obtained from abscess cultures confirm the polymicrobial nature of supportive infections, as demonstrated by the disparity found in the literature. For example our data highlight the isolation of two or more species of facultative anaerobes and/or obligate anaerobic, often including F. necrophorum and Bacteroides spp. The most common conditions in which F. necrophorum participates as a primary agent are liver abscesses in conjunction with A. pyogenes , Dichelobacter spp ., Staphylococcus spp. and Streptococcus spp
Fusobacterium spp. and Dichelobacter spp. constitute more than half of anaerobes isolated from mixed opportunistic infections. However, the difficulties in their isolation and identification can lead to an underestimation of their involvement. This may have occurred in the present study because the method employed for detection of these bacteria was not sufficiently specific for their complete identification.
Our results do not completely agree with other studies, in which a greater variety of pathogens and saprophytes were isolated from abscesses: A. pyogenes , E. coli, Enterococcus spp., Enterobacter spp ., P. aeruginosa, Bacteroides spp., Aeromonas hydrophila, Citrobacter spp., and several Staphylococci spp. This is likely due to differences in the culture methods and bacteriological techniques used. It is also possible that nutritional pathologies such as selenium deficiency and/or vitamin E deficiency that produce lipid hepatosis may have influenced the inability to identify certain agents and the existence of cultures without growth.
Azizi et al. [43] reported a similar percentage of abscesses in adult sheep lungs. The origin of lung abscesses is complicated to establish because they may be secondary to other unresolved processes and contaminated with pyogenic germs or because the pyogenic germs may have ended up in pulmonary tissue and formed abscesses, as appears to be the most common case in the liver.
With the techniques used in this study, it would be exceptionally difficult to determine the cause of liver or lung abscesses, and it would be even more difficult using routine slaughterhouse examination. Therefore, it would be necessary to resort to more sophisticated laboratory techniques to obtain a more accurate assessment.
Caseous lymphadenitis
The difficulties associated with the complexity of current inspection procedures are evident, and the inspection criteria used to diagnose caseous lymphadenitis should be re-evaluated. In our study and as noted the growth of C. pseudo tuberculosis in suspicious lesions was very useful for this diagnosis because the lesions occasionally did not show a clear macroscopic pattern and could be confused with abscesses caused by other germs. More recently, Zavosthi et al. [38] argued that microscopic examination presented a more precise diagnosis than did bacteriological culture and macroscopic evaluation of lymph nodes. Thus, the annual prevalence of caseous lymphadenitis may be higher than the rate we determined in adult sheep. Nevertheless, our rate is within the range found by some post-mortem inspections in the slaughterhouse studies.
The seasons with the highest and lowest incidence of this disease were spring (11.91%), and autumn (1.11%) (p<0.05). Some authors do not consider real seasonality but additional factors (e.g., the mixing of different breeds, presence of mixed sheep, and the attendance of sheep to livestock fairs, methods of restraint used during the shearing) could be the primary influence on the epidemiology of the disease. Thus, the seasonal differences found in the present study may be due to sheep shearing that occurs in most of Spain during late winter and spring. The use of unhygienic shearing techniques without adequate disinfection of the material used between individuals may be a contributing factor. This sense indicated that the high sero-prevalence of caseous lymphadenitis that they found in a Brazilian abattoir was related to the lack of good management measures on the farms.
C. pseudo tuberculosis was isolated in all caseous lymphadenitis lesions, with S. aureus or other pathogens occasionally growing concomitantly. These results are in agreement with other studies that reported that both species were the most frequent isolates in caseous lymphadenitis lesions.
Only one case of abscess disease was identified in association to caseous lymphadenitis so, it did not appear to be relevant but rather a fortuitous circumstance. One may argue that the growth of both pathogens from the same lesion was due to the secondary contamination of the abscess, although it is very difficult to determine which pathogen grew first in the lesion and generated the disease. Abscess disease has proven to be an uncommon finding and complicates the diagnosis during post-mortem inspection because it presents no characteristic lesion beyond a purulent abscess. The obtained data is in contrast to the findings in Rasa Aragonesa sheep in Aragon, which found abscess disease prevalence of 60-70% of animals.

Conclusion

Enzootic pneumonia in lambs and sheep was by far the most relevant bacterial pathology in the ovine slaughterhouse under study. The season appeared to be an important variable in the generation of ovine rejections for the majority of the diseases identified; the greatest losses in lambs occurred in the spring, whereas in sheep, they occurred in the spring and summer.
The results clearly show that the vast majority of the ovine bacterial pathologies are caused by a handful of bacterial species: M. haemolytica , P. multocida , A. pyogenes , C. pseudo tuberculosis and S. aureus. Also, this study points out the relevance of Mycoplasma spp. as a major component of the enzootic pneumonia complex although its investigation requires very specific and costly techniques.

Acknowledgments

This study was supported by the Spanish Research Project RTA2010-00094-C03-03.

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