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Salmonella enterica subsp. diarizonae serotype 61:k:1,5,(7) associated with chronic proliferative rhinitis and high nasal colonization rates in a flock of Texel sheep in Switzerland
Preventive Veterinary Medicine 145 (2017) 78–82
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Salmonella enterica subsp. diarizonae serotype 61:k:1,5,(7) associated with chronic proliferative rhinitis and high nasal colonization rates in a flock of Texel sheep in Switzerland Nadine Stokar-Regenscheit a,∗,1 , Gudrun Overesch b,1 , Regula Giezendanner c , Simone Roos a,2 , Corinne Gurtner a a
Institute of Animal Pathology, Vetsuisse Faculty, University of Bern, Länggassstrasse 122, 3001 Bern, Switzerland Institute of Veterinary Bacteriology, Vetsuisse Faculty, University of Bern, Länggassstrasse 122, 3001 Bern, Switzerland c Swiss Health Service for Small Ruminants, Postfach, 3360 Herzogenbuchsee, Switzerland b
a r t i c l e
i n f o
Article history: Received 28 November 2016 Received in revised form 19 June 2017 Accepted 2 July 2017 Keywords: Salmonella Nasal discharge Nasal carriers Mucosal proliferation Zoonotic potential
a b s t r a c t Salmonella (S.) enterica subspecies diarizonae (IIIb) serovar 61:(k):1,5,(7) (S. IIIb 61:(k):1,5,(7)) is considered to be host adapted to sheep and is found regularly in feces of healthy carriers and of sheep with salmonellosis. A few cases of chronic proliferative rhinitis (CPR) in sheep have been described as a new disease in association with S. IIIb 61:k:1,5,(7) in the USA, in Spain and now for the first time in Switzerland. Three animals of a flock of Texel sheep suffering from chronic nasal discharge and dyspnea with subsequent death were necropsied. The pathological lesions are consistent with a severe proliferation of the nasal mucosae of the turbinates in association with severe chronic inflammation. S. IIIb 61:(k):1,5,(7) was isolated from the lesions by direct bacteriological culture and the presence of Salmonella spp. was confirmed by immunohistochemistry. The affected flock was systematically tested after the first occurrence of the disease. Clinical examination of the flock revealed approx. 20% of the adult sheep to show nasal discharge, approx. 5% having severe dyspnea and approx. 5% having chronic intermittent diarrhea. Lambs (n = 28) showed no clinical signs at all. High positivity of nasal mucosa (46%), but low prevalence in feces (6%) for S. IIIb 61:k:1,5,(7) was found. The results lead to the assumption of a direct animal to animal transmission by nasal discharge followed by a chronic disease leading to death after several months to years. Animals tested positive for S. IIIb 61:k:1,5,(7) were all >1 year old. CPR represents a chronic disease in adult sheep posing a risk for spreading S. IIIb 61:k:1,5,(7) between flocks and with a zoonotic potential. © 2017 Published by Elsevier B.V.
1. Introduction Salmonella (S.) enterica subspecies diarizonae serovar 61:k:1,5,(7), also known as subspecies IIIb (Le Minor et al., 1982) (S. IIIb 61:(k):1,5,(7)), was first isolated from reptiles in 1939 (Caldwell and Ryerson, 1939) and 1952 found in carcasses of aborted ovine fetuses (Ryff and Brown, 1952). Today, S. IIIb 61:(k):1,5,(7) is con-
∗ Corresponding author. E-mail addresses: [email protected] (N. Stokar-Regenscheit), [email protected] (G. Overesch), [email protected] (R. Giezendanner), [email protected] (C. Gurtner). 1 These two authors contributed equally to this study. 2 Present address: Institute of Pathology and Forensic Veterinary Medicine, Unit of Laboratory Animal Pathology, University of Veterinary Medicine, Veterinärplatz 1, 1210 Vienna, Austria. http://dx.doi.org/10.1016/j.prevetmed.2017.07.003 0167-5877/© 2017 Published by Elsevier B.V.
sidered host adapted to sheep and colonizes the ovine intestine and tonsils without clinical signs and pathological findings (Weiss et al., 1986; Hall and Rowe, 1992; Sanberg et al., 2002; Bonke et al., 2012). In Sweden, S. IIIb 61:(k):1,5,(7) is considered to be endemic in sheep herds (Sörén et al., 2015). Dargatz et al. (2015) noted a high prevalence of Salmonella spp. in ovine fecal samples, with 72% of tested farms having at least one positive sample. 94% of these Salmonella isolates were identified as S. IIIb 61:(k):1,5,(7). Isolation of S. IIIb 61:(k):1,5,(7) has been sporadically reported from a variety of clinical disorders in sheep in Canada, Europe and the USA: Diarrhea in lambs (Harp et al., 1981), abortions (Long et al., 1978; Pritchard, 1990; Davies et al., 2001), a case of unilateral suppurative epididymo-orchitis in a 2-year-old ram (Ferreras et al., 2007) and cases of chronic proliferative rhinitis (Meehan et al., 1992; Lacastra et al., 2012).
N. Stokar-Regenscheit et al. / Preventive Veterinary Medicine 145 (2017) 78–82
In 2001, an increased isolation of S. IIIb 61:(k):1,5,(7) from ovine samples submitted to veterinary laboratories in the UK was detected, which is in contrast to a substantial reduction of Salmonella in all livestock species (Davies et al., 2001). Reports from Norway refer to high regional differences in the prevalence of S. IIIb 61:(k):1,5,(7) in fecal samples ranging from 0 to 45% (Alvseike and Skjerve, 2002). Cecum samples collected from 653 slaughtered sheep from abattoirs in 2002 in Switzerland showed a prevalence of 11% for S. IIIb 61:k:1,5,(7), indicating sheep are a reservoir for this serovar (Zweifel et al., 2004). In the UK, the overall prevalence of Salmonella spp. from feces of slaughtered sheep was low (1.1%), but S. IIIb 61:(k):1,5,(7) was the most frequent Salmonella serotype isolated with seasonal spring period being of highest risk (Milnes et al., 2009). In Norway and Sweden, low within-herd prevalences of rectal samples from 14% and 17.6%, respectively were reported (Sanberg et al., 2002; Sörén et al., 2015). Interestingly, there were marked differences between large farms (40%) and small farms (12%), but with an equal distribution in all counties (Sörén et al., 2015). As a consequence of this result, the responsible authorities in Sweden excluded S. IIIb 61:(k):1,5,(7) from regular control programs on Salmonella in sheep (Sören et al., 2015). Dargatz et al. (2015) concluded in their investigation from 2011, that salmonellae typically associated with foodborne disease transmission are infrequently found on sheep farms in the USA. Chronic proliferative rhinitis (CPR) associated with S. IIIb 61:k:1,5,(7) was first described 1992 in two cases (Columbian and Dorset sheep) in the USA (Meehan et al., 1992). More recently, S. IIIb 61:(k):1,5,(7) was isolated from sheep with CPR in several flocks of Aragonasa sheep in Spain (Lacastra et al., 2012). An experimental reproduction of CPR was not fully successful and nasally instilled S. IIIb 61:(k):1,5,(7) could persist for several months in the nasal mucosa of lambs, but only induce a mild proliferative response (Brodgen et al., 1994). No data on nasal isolation of S. IIIb 61:k:1,5,(7) in clinically healthy sheep are available. This study is the first report of a systematic monitoring of nasal and fecal swabs of a flock of sheep showing nasal discharge and CPR associated with S. IIIb 61:k:1,5,(7). It gives new insight into the pathogenesis of a possibly, so far, underestimated entity of salmonellosis. This knowledge is important for official authorities to consider S. IIIb 61:k:1,5,(7) associated with CPR as a clinically relevant form of salmonellosis in sheep with zoonotic potential.
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(HE). Immunohistochemistry for Salmonella spp. was performed by Dr. De las Heras, Departamento de Patologia Animal, Universidad de Zaragoza in Spain as described elsewhere (Lacastra et al., 2012). 2.2. Sampling of the remaining Texel flock All remaining sheep of the flock (n = 59) were clinically examined in July 2015, three months after the first isolation of Salmonella spp. from the nasal turbinates of the necropsied animal no. 2. Of these sheep, 28 were lambs (9 males, 19 female), born in the same year in spring and 31 animals (5 males, 26 females) were adults (> one year old). The ewes were in their first or up to the 9th lactation period. Of these sheep, nasal swabs (n = 59) and fecal samples (n = 51) were taken for bacteriological culture. 2.3. Bacteriological cultures Tissue of affected nasal turbinates from two animals (no. 2 and 3) were analysed for Salmonella spp. by direct detection on selective agar plates (Brilliant Green agar and Brilliance Salmonella agar, Oxoid Ltd., Basingstoke, UK) incubated at 37 ◦ C ± 1 ◦ C for 24 h ± 3 h. Colonies suspicious for Salmonella spp. on selective agar plates were sub-cultured on trypticase soy agar plates with 5% sheep blood at 37 ◦ C ± 1 ◦ C for 24 h ± 3 h (Becton Dickinson, Franklin Lakes, NJ, USA). Salmonella spp. was confirmed by matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy (MALDI TOF MS) using the direct transfer method (Biotyper 3.0, Bruker Daltonics, Bremen, Germany). Salmonella spp. was identified to species and subspecies level using routine biochemical methods; the serovar was assigned by serotyping according to the International Organization for Standardization (ISO) 6579:2014–Part 3: Horizontal method for the detection, enumeration and serotyping of Salmonella − Part 3: Guidelines for serotyping of Salmonella spp. Nasal swabs and feces were cultivated in Muller-Kauffmann Tetrathionate-Novobiocin Broth (MKTTn, Oxoid ltd.) 37 ◦ C ± 1 ◦ C for 24 h ± 3 h. Incubated MKTTn broth were sub-cultured onto two Salmonella selective agar plates (Brilliant Green agar and Brilliance Salmonella agar (Oxoid Ltd.)) and incubated for further 24 h ± 3 h at 37 ◦ C ± 1 ◦ C. Suspicious colonies were processed as described above. 3. Results
2. Materials and methods 3.1. Clinical findings 2.1. Post mortem pathology analysis Three adult Texel sheep (animal no. 1, male intact, 4.5 years; animal no. 2, female intact, 5.5 years; animal no. 3, female intact, 5.5 years) of the same flock were submitted for necropsy in 2014 and 2015 (Table 1). A complete necropsy of two animals (no. 1 and no. 2) was performed, while only the head of animal no. 3 was examined. Gross lesions were described and photographically documented. Fresh tissue samples of the affected nasal turbinates were immediately fixed in 4% neutral buffered formalin and embedded in paraffin. Sections of 4 m were stained with hematoxylin and eosin
All three necropsied animals showed chronic dyspnea, respiratory distress and nasal discharge, with severity slowly increasing over several months (Table 1). They had obvious deformation of the nasal bridge and proliferative tissue was visible at the nares (Fig. 1A). Ram no. 1 was euthanized in extremis with severe dyspnea. Ewes no. 2 and 3 were found dead after a chronic episode of nasal discharge and dyspnea. Ram no. 1 was imported from a Texel breeder in Germany in 2010 and had not been tested for Salmonella spp. in quarantine during import procedures, as no clinical signs of diarrhea were present. The ram was the first animal in the flock
Table 1 Bacteriological and clinical findings in Texel sheep dying/euthanized because of chronic proliferative rhinitis in Switzerland in 2014 and 2015. animal no.
1 2 3
age (years)
4.5 5.5 5.5
sex
ram ewe ewe
nd; not done. a for S. enterica subspecies diarizonae serotype 61:k:1,5,(7).
bacteriological culture
clinical signs
nasal swab
feces
nasal
feces
nd posa posa
nd nd nd
dyspnea, nasal discharge dyspnea, nasal discharge dyspnea, nasal discharge
none none none
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Fig 1. Gross and histopathological lesions of chronic proliferative rhinitis (CPR) of Texel sheep no. 2. (A) An obvious deformation of the nasal bridge (arrow head) and the extruding mucosal proliferation is visible at the nares (long arrow). (B) Sagittal cut section of the head: The conchal mucosa is diffusely, severely thickened and obstructing the nasal space (stars). (C) Histology of the nasal mucosa: The submucosa is severely infiltrated by lymphocytes and plasma cells (asterisk). There is a severe, diffuse hyperplasia of epithelial and goblet cells (arrows). The epithelial cell layer is diffusely covered with large amounts of mucous and exfoliated cells as well as degenerated neutrophils (triangle). (HE, 100×). (D) Immunohistochemistry for Salmonella spp. shows positive immunostaining within inflamed areas. (IHC Salmonella spp., 400×).
showing signs of nasal discharge, dyspnea, and respiratory distress in 2014 at 4 years of age. With 4.5 years he was euthanized due to his poor condition and poor prognosis. Clinical examination of the remaining sheep (n = 59) from the affected flock revealed approx. 12 adult sheep to show nasal discharge (20%), approx. 3 having severe dyspnea (5%) and approx. another 3 having chronic intermittent diarrhea (5%). Lambs (n = 28) showed no clinical signs at all. Otherwise the flock was considered healthy and no other diseases were reported. The breeder took part in a parasite surveillance program of the Swiss small ruminant consultant association and therefore all animals had been dewormed 2–3 times per year alternating with ® ® Cydectin (Zoetis Switzerland GmbH), Endex (Novartis animal ® health, Switzerland) and Hepadex (MSD Animal Health GmbH) accompanied by an efficacy fecal parasitological monitoring. 3.2. Pathology In the post mortem examination, the mucosa of the nasal turbinates showed a bilateral symmetric, severe thickening up to
1 cm, which completely obstructed the nasal airways reaching from the nares to the naso-pharynx (Fig. 1B). The mucosa was reddish to purple, had a smooth surface and was covered with a large amount of viscous, white-grey, turbid mucus. All three animals showed a severe, diffuse, acute pulmonary edema. Histologically, the mucosa and submucosa of the nasal turbinates were severely expanded and infiltrated by mainly lymphocytes, plasma cells and less neutrophils and macrophages. Often Mott cells were present. There was severe, diffuse hyperplasia of epithelial and goblet cells (Fig. 1C). The epithelial cell layer was diffusely covered with large amounts of mucous and exfoliated cells as well as degenerated neutrophils. There were multifocal areas of necrosis and accumulations of degranulated eosinophils. Within the submucosa were multiple areas of pronounced granulation tissue. The underlying cartilage of the nasal turbinates showed moderate chondrolysis and remodeling. By immunohistochemistry, Salmonella spp. antigen was detected in large amounts within inflamed areas extraand intracellularly (Fig. 1D).
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Table 2 Results of Salmonella enterica subsp. diarizonae serotype 61:k:1,5,(7) culture of the flock of Texel sheep (n = 59) from nasal swabs and feces differentiating between age and sex. number of animals (n)
28 31 a b
sex
age
9 19 5 26
a
juvenile (lambs) juvenile (lambs)a adultb adultb
male female male (rams) female (ewes)
number of Salmonella positive samples (n) nasal swabs
feces
0 0 4 23
0 0 0 3
lambs born in spring in the same year of sampling (July 2015). >1 year old.
3.3. Bacteriology Subsequent bacteriological culture of the nasal turbinates after post mortem examination of both ewes (no. 2 and 3) revealed presence of S. IIIb 61:k:1,5,(7) (Table 1). Of the 59 investigated nasal swabs from the remaining flock, all lambs (n = 28) were negative by enrichment for S. IIIb 61:k:1,5,(7). From the 31 adults 87% (n = 27) were positive for S. IIIb 61:k:1,5,(7). From 51 fecal samples, three (6%) samples from adults were positive by enrichment for S. IIIb 61:k:1,5,(7) (Table 2). All three animals positive in the fecal samples were positive in the nasal swabs as well. The ewes in their first lactation yielded positive nasal swabs and were tested negative in the feces. All negative tested animals in the nasal swabs were also negative in the feces. 4. Discussion Three Texel sheep from a flock of 62 sheep in Switzerland either died or were euthanized due to severe illness caused by CPR in association with S. IIIb 61:k:1,5,(7). This is the first report describing CPR with isolation of S. IIIb 61:k:1,5,(7) in Switzerland, the third country worldwide, beside the USA and Spain (Meehan et al., 1992; Lacastra et al., 2012), reporting this disease. S. IIIb 61:k:1,5,(7), from the inflamed nasal mucosa, was isolated by direct bacteriological culture without enrichment, indicating a high bacterial load of Salmonella spp. Salmonella spp. antigen was co-localized within the lesions by immunohistochemistry. In previous cases in the USA, positive immuno-gold labeling for Salmonella spp. of intracellular bacilli in goblet cells within host cell membrane-bound organelles was identified by transmission electron micrograph (Meehan et al., 1992). The detection of Salmonella spp. antigen within the lesion leads to the strong assumption that S. IIIb 61:k:1,5,(7) induces CPR in sheep. In the present report, three animals with CPR were clinically ill, exhibiting chronic nasal discharge, dyspnea and respiratory distress leading to death. The examined animals suffered from these clinical signs for several months to years, which is consistent with the previously reported chronic nature of the disease (Meehan et al., 1992; Lacastra et al., 2012). Bacteriological analysis of the remaining sheep (n = 59) in the flock revealed a high nasal colonization rate with 87%of the adult sheep (n = 31) positiv. Approx. ½ (n = 15) of these colonized sheep showed clinical signs of the upper respiratory tract. Interestingly, all lambs (<6 months, n = 28) of the flock were negative while 23 of 26 of their mothers (89%) were tested positive in the nasal swabs. This shows that the nasal colonization of sheep with this specific Salmonella serovar takes several months up to a year, even if the animals are in close contact. Possible explanations are: 1) lambs are protected by maternal antibodies; 2) to become infected, animals need a time of immunosuppression/an immunosuppressive period (e.g. pregnancy, stress); 3) previous infection or co-infection by another organism (unknown) is needed. Further investigations
on weaning lambs are necessary to clarify at what time point sheep acquire colonization and infection of S. IIIb 61:k:1,5,(7). Our data leads to the assumption, that lambs can be separated from their mothers, and the remaining adult sheep of the flock, without infection during their first year in order to eradicate Salmonella in the flock. This needs further investigation, but will be an important input for authorities in case of eradication. Our findings, that lambs are not infected in the first year are supported by data of other countries, where in one case the majority of the positively tested animals were older than two years (Sanberg et al., 2002) and in another case they were older than 6 months (at slaughter) (Milnes et al., 2009). It is discussed, that S. IIIb 61:k:1,5,(7) becomes invasive if animals are stressed or immunocompromised (Greenfield and Greenway, 1973; Harp et al., 1981; Pritchard, 1990). In Norway, different stressors such as mating, vaccination and late pregnancy did not seem to influence the prevalence of S. IIIb 61:k:1,5,(7) in fecal samples (Sanberg et al., 2002). Stress may have been associated with three cases of CPR found in one of the flocks described in Spain, as the disease occurred shortly after vaccination (Lacastra et al., 2012). The most evident risk factors on herd level described in Norway were above-median herd size, having white crossbreed sheep, and higher annual replacement percentages (Alvseike and Skjerve, 2002). The outbreak of the respiratory signs and the disease of CPR in the flock started with the import of a clinically healthy ram from Germany. After introduction of the ram into the flock in 2010, several sheep of the flock apart from the ram started showing respiratory signs, suggesting S. IIIb 61:k:1,5,(7) was introduced and further transmitted by the ram to other animals in the flock. This might present a neglected way of shedding bacteria by nasal secretion or even a new path of transmission in the salmonellosis pathogenesis. The transport of the ram may have led to high stress levels and to shedding of S. IIIb 61:k:1,5,(7) leading to a transmission to other sheep in the new flock possibly by nasal discharge. Furthermore, the introduction of the new ram may have been a stressor for the remaining sheep facilitating transmission of S. IIIb 61:k:1,5,(7) from animal to animal within the flock. This is in contrast to the report by Sanberg et al. (2002) in Norway, where sharing of rams between flocks did not seem to be a risk factor for the presence of S. IIIb 61:k:1,5,(7). Furthermore, in that study, authors observed no association between prevalence and participation in ram circles; therefore, they concluded that the infection is not transmitted from animal to animal (Sanberg et al., 2002). However, the results in the present investigation strongly indicate a direct transmission from animal to animal within flocks. A representative monitoring on sheep flock is needed to evaluate the nasal prevalence of S. IIIb 61:k:1,5,(7) in both clinically healthy and CPR affected sheep. So far the epidemiology of CPR including the route of transmission and zoonotic risk analysis have not been investigated. Detailed analyses of the clinical status of sheep are mandatory to differentiate between colonization and infection.
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Through such studies, the economic impact on ovine meat and milk industry might be estimated more reliably. However, the pathogen has zoonotic potential and human infections with this distinct serovar are reported worldwide (Hall and Rowe (1992); Weiss et al. (1986); Sirmatel et al. (1998); Hjartardóttir et al., 2012). Human infections occur predominantly via the foodborne route and findings of S. IIIb 61:k:1,5,(7) in livestock are therefore of human health importance (Weiss et al., 1986; Hall and Rowe, 1992). In humans S. IIIb 61:k:1,5,(7) is among other Salmonella serotypes described as a more invasive serotype which is associated with a higher mortality rate than the low invasive serotypes (Weiss et al., 1986). Although human infections with S. IIIb 61:k:1,5,(7) rarely occur, the possible critical outcome for humans makes it necessary to pay attention and keep sheep as an important reservoir of this zoonotic organism in mind. 5. Conclusion High nasal detection rates of S. IIIb 61:k:1,5,(7) in clinically affected Texel sheep are associated with three cases of CPR. We assume that nasal colonization of S. IIIb 61:k:1,5,(7) in adults may lead to chronic nasal infections with respiratory signs and lethal mucosal proliferation in the nose. The investigation of the associated flock indicates a direct animal to animal transmission and a possible new way of shedding Salmonella by nasal discharge. Monitoring of sheep flock for nasal colonization including clinical examination is needed to understand the importance of S. IIIb 61:k:1,5,(7) in sheep production and its zoonotic risk. Furthermore, the role of carrier animals will have to be further analyzed and discussed by the official authorities. Conflicts of interest None. Funding No external funding was used for the project. Acknowledgements We thank the owner of the flock of Texel sheep Mr. Bieri, for providing us with information on the flock and giving us access to his sheep for sampling. The veterinary students Yannik Fussen and Nadine Zimmermann were very enthusiastic about sampling and we thank them for their great help with our field sampling. Further on, we thank Stefanie Müller and Isabelle Brodard for their excellent technical assistance in the bacteriological analyses, as well as Manuela Bozzo and Erika Bürgi for the histology slide preparation. Many thanks go to Dr. De las Heras, Departamento de Patologia Animal, Universidad de Zaragoza, Spain for performing the Salmonella spp. immunohistochemistry.
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Salmonella enterica subsp. diarizonae serotype 61:k:1,5,(7) associated with chronic proliferative rhinitis and high nasal colonization rates in a flock of Texel sheep in Switzerland Nadine Stokar-Regenscheit a,∗,1 , Gudrun Overesch b,1 , Regula Giezendanner c , Simone Roos a,2 , Corinne Gurtner a a
Institute of Animal Pathology, Vetsuisse Faculty, University of Bern, Länggassstrasse 122, 3001 Bern, Switzerland Institute of Veterinary Bacteriology, Vetsuisse Faculty, University of Bern, Länggassstrasse 122, 3001 Bern, Switzerland c Swiss Health Service for Small Ruminants, Postfach, 3360 Herzogenbuchsee, Switzerland b
a r t i c l e
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Article history: Received 28 November 2016 Received in revised form 19 June 2017 Accepted 2 July 2017 Keywords: Salmonella Nasal discharge Nasal carriers Mucosal proliferation Zoonotic potential
a b s t r a c t Salmonella (S.) enterica subspecies diarizonae (IIIb) serovar 61:(k):1,5,(7) (S. IIIb 61:(k):1,5,(7)) is considered to be host adapted to sheep and is found regularly in feces of healthy carriers and of sheep with salmonellosis. A few cases of chronic proliferative rhinitis (CPR) in sheep have been described as a new disease in association with S. IIIb 61:k:1,5,(7) in the USA, in Spain and now for the first time in Switzerland. Three animals of a flock of Texel sheep suffering from chronic nasal discharge and dyspnea with subsequent death were necropsied. The pathological lesions are consistent with a severe proliferation of the nasal mucosae of the turbinates in association with severe chronic inflammation. S. IIIb 61:(k):1,5,(7) was isolated from the lesions by direct bacteriological culture and the presence of Salmonella spp. was confirmed by immunohistochemistry. The affected flock was systematically tested after the first occurrence of the disease. Clinical examination of the flock revealed approx. 20% of the adult sheep to show nasal discharge, approx. 5% having severe dyspnea and approx. 5% having chronic intermittent diarrhea. Lambs (n = 28) showed no clinical signs at all. High positivity of nasal mucosa (46%), but low prevalence in feces (6%) for S. IIIb 61:k:1,5,(7) was found. The results lead to the assumption of a direct animal to animal transmission by nasal discharge followed by a chronic disease leading to death after several months to years. Animals tested positive for S. IIIb 61:k:1,5,(7) were all >1 year old. CPR represents a chronic disease in adult sheep posing a risk for spreading S. IIIb 61:k:1,5,(7) between flocks and with a zoonotic potential. © 2017 Published by Elsevier B.V.
1. Introduction Salmonella (S.) enterica subspecies diarizonae serovar 61:k:1,5,(7), also known as subspecies IIIb (Le Minor et al., 1982) (S. IIIb 61:(k):1,5,(7)), was first isolated from reptiles in 1939 (Caldwell and Ryerson, 1939) and 1952 found in carcasses of aborted ovine fetuses (Ryff and Brown, 1952). Today, S. IIIb 61:(k):1,5,(7) is con-
∗ Corresponding author. E-mail addresses: [email protected] (N. Stokar-Regenscheit), [email protected] (G. Overesch), [email protected] (R. Giezendanner), [email protected] (C. Gurtner). 1 These two authors contributed equally to this study. 2 Present address: Institute of Pathology and Forensic Veterinary Medicine, Unit of Laboratory Animal Pathology, University of Veterinary Medicine, Veterinärplatz 1, 1210 Vienna, Austria. http://dx.doi.org/10.1016/j.prevetmed.2017.07.003 0167-5877/© 2017 Published by Elsevier B.V.
sidered host adapted to sheep and colonizes the ovine intestine and tonsils without clinical signs and pathological findings (Weiss et al., 1986; Hall and Rowe, 1992; Sanberg et al., 2002; Bonke et al., 2012). In Sweden, S. IIIb 61:(k):1,5,(7) is considered to be endemic in sheep herds (Sörén et al., 2015). Dargatz et al. (2015) noted a high prevalence of Salmonella spp. in ovine fecal samples, with 72% of tested farms having at least one positive sample. 94% of these Salmonella isolates were identified as S. IIIb 61:(k):1,5,(7). Isolation of S. IIIb 61:(k):1,5,(7) has been sporadically reported from a variety of clinical disorders in sheep in Canada, Europe and the USA: Diarrhea in lambs (Harp et al., 1981), abortions (Long et al., 1978; Pritchard, 1990; Davies et al., 2001), a case of unilateral suppurative epididymo-orchitis in a 2-year-old ram (Ferreras et al., 2007) and cases of chronic proliferative rhinitis (Meehan et al., 1992; Lacastra et al., 2012).
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In 2001, an increased isolation of S. IIIb 61:(k):1,5,(7) from ovine samples submitted to veterinary laboratories in the UK was detected, which is in contrast to a substantial reduction of Salmonella in all livestock species (Davies et al., 2001). Reports from Norway refer to high regional differences in the prevalence of S. IIIb 61:(k):1,5,(7) in fecal samples ranging from 0 to 45% (Alvseike and Skjerve, 2002). Cecum samples collected from 653 slaughtered sheep from abattoirs in 2002 in Switzerland showed a prevalence of 11% for S. IIIb 61:k:1,5,(7), indicating sheep are a reservoir for this serovar (Zweifel et al., 2004). In the UK, the overall prevalence of Salmonella spp. from feces of slaughtered sheep was low (1.1%), but S. IIIb 61:(k):1,5,(7) was the most frequent Salmonella serotype isolated with seasonal spring period being of highest risk (Milnes et al., 2009). In Norway and Sweden, low within-herd prevalences of rectal samples from 14% and 17.6%, respectively were reported (Sanberg et al., 2002; Sörén et al., 2015). Interestingly, there were marked differences between large farms (40%) and small farms (12%), but with an equal distribution in all counties (Sörén et al., 2015). As a consequence of this result, the responsible authorities in Sweden excluded S. IIIb 61:(k):1,5,(7) from regular control programs on Salmonella in sheep (Sören et al., 2015). Dargatz et al. (2015) concluded in their investigation from 2011, that salmonellae typically associated with foodborne disease transmission are infrequently found on sheep farms in the USA. Chronic proliferative rhinitis (CPR) associated with S. IIIb 61:k:1,5,(7) was first described 1992 in two cases (Columbian and Dorset sheep) in the USA (Meehan et al., 1992). More recently, S. IIIb 61:(k):1,5,(7) was isolated from sheep with CPR in several flocks of Aragonasa sheep in Spain (Lacastra et al., 2012). An experimental reproduction of CPR was not fully successful and nasally instilled S. IIIb 61:(k):1,5,(7) could persist for several months in the nasal mucosa of lambs, but only induce a mild proliferative response (Brodgen et al., 1994). No data on nasal isolation of S. IIIb 61:k:1,5,(7) in clinically healthy sheep are available. This study is the first report of a systematic monitoring of nasal and fecal swabs of a flock of sheep showing nasal discharge and CPR associated with S. IIIb 61:k:1,5,(7). It gives new insight into the pathogenesis of a possibly, so far, underestimated entity of salmonellosis. This knowledge is important for official authorities to consider S. IIIb 61:k:1,5,(7) associated with CPR as a clinically relevant form of salmonellosis in sheep with zoonotic potential.
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(HE). Immunohistochemistry for Salmonella spp. was performed by Dr. De las Heras, Departamento de Patologia Animal, Universidad de Zaragoza in Spain as described elsewhere (Lacastra et al., 2012). 2.2. Sampling of the remaining Texel flock All remaining sheep of the flock (n = 59) were clinically examined in July 2015, three months after the first isolation of Salmonella spp. from the nasal turbinates of the necropsied animal no. 2. Of these sheep, 28 were lambs (9 males, 19 female), born in the same year in spring and 31 animals (5 males, 26 females) were adults (> one year old). The ewes were in their first or up to the 9th lactation period. Of these sheep, nasal swabs (n = 59) and fecal samples (n = 51) were taken for bacteriological culture. 2.3. Bacteriological cultures Tissue of affected nasal turbinates from two animals (no. 2 and 3) were analysed for Salmonella spp. by direct detection on selective agar plates (Brilliant Green agar and Brilliance Salmonella agar, Oxoid Ltd., Basingstoke, UK) incubated at 37 ◦ C ± 1 ◦ C for 24 h ± 3 h. Colonies suspicious for Salmonella spp. on selective agar plates were sub-cultured on trypticase soy agar plates with 5% sheep blood at 37 ◦ C ± 1 ◦ C for 24 h ± 3 h (Becton Dickinson, Franklin Lakes, NJ, USA). Salmonella spp. was confirmed by matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy (MALDI TOF MS) using the direct transfer method (Biotyper 3.0, Bruker Daltonics, Bremen, Germany). Salmonella spp. was identified to species and subspecies level using routine biochemical methods; the serovar was assigned by serotyping according to the International Organization for Standardization (ISO) 6579:2014–Part 3: Horizontal method for the detection, enumeration and serotyping of Salmonella − Part 3: Guidelines for serotyping of Salmonella spp. Nasal swabs and feces were cultivated in Muller-Kauffmann Tetrathionate-Novobiocin Broth (MKTTn, Oxoid ltd.) 37 ◦ C ± 1 ◦ C for 24 h ± 3 h. Incubated MKTTn broth were sub-cultured onto two Salmonella selective agar plates (Brilliant Green agar and Brilliance Salmonella agar (Oxoid Ltd.)) and incubated for further 24 h ± 3 h at 37 ◦ C ± 1 ◦ C. Suspicious colonies were processed as described above. 3. Results
2. Materials and methods 3.1. Clinical findings 2.1. Post mortem pathology analysis Three adult Texel sheep (animal no. 1, male intact, 4.5 years; animal no. 2, female intact, 5.5 years; animal no. 3, female intact, 5.5 years) of the same flock were submitted for necropsy in 2014 and 2015 (Table 1). A complete necropsy of two animals (no. 1 and no. 2) was performed, while only the head of animal no. 3 was examined. Gross lesions were described and photographically documented. Fresh tissue samples of the affected nasal turbinates were immediately fixed in 4% neutral buffered formalin and embedded in paraffin. Sections of 4 m were stained with hematoxylin and eosin
All three necropsied animals showed chronic dyspnea, respiratory distress and nasal discharge, with severity slowly increasing over several months (Table 1). They had obvious deformation of the nasal bridge and proliferative tissue was visible at the nares (Fig. 1A). Ram no. 1 was euthanized in extremis with severe dyspnea. Ewes no. 2 and 3 were found dead after a chronic episode of nasal discharge and dyspnea. Ram no. 1 was imported from a Texel breeder in Germany in 2010 and had not been tested for Salmonella spp. in quarantine during import procedures, as no clinical signs of diarrhea were present. The ram was the first animal in the flock
Table 1 Bacteriological and clinical findings in Texel sheep dying/euthanized because of chronic proliferative rhinitis in Switzerland in 2014 and 2015. animal no.
1 2 3
age (years)
4.5 5.5 5.5
sex
ram ewe ewe
nd; not done. a for S. enterica subspecies diarizonae serotype 61:k:1,5,(7).
bacteriological culture
clinical signs
nasal swab
feces
nasal
feces
nd posa posa
nd nd nd
dyspnea, nasal discharge dyspnea, nasal discharge dyspnea, nasal discharge
none none none
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Fig 1. Gross and histopathological lesions of chronic proliferative rhinitis (CPR) of Texel sheep no. 2. (A) An obvious deformation of the nasal bridge (arrow head) and the extruding mucosal proliferation is visible at the nares (long arrow). (B) Sagittal cut section of the head: The conchal mucosa is diffusely, severely thickened and obstructing the nasal space (stars). (C) Histology of the nasal mucosa: The submucosa is severely infiltrated by lymphocytes and plasma cells (asterisk). There is a severe, diffuse hyperplasia of epithelial and goblet cells (arrows). The epithelial cell layer is diffusely covered with large amounts of mucous and exfoliated cells as well as degenerated neutrophils (triangle). (HE, 100×). (D) Immunohistochemistry for Salmonella spp. shows positive immunostaining within inflamed areas. (IHC Salmonella spp., 400×).
showing signs of nasal discharge, dyspnea, and respiratory distress in 2014 at 4 years of age. With 4.5 years he was euthanized due to his poor condition and poor prognosis. Clinical examination of the remaining sheep (n = 59) from the affected flock revealed approx. 12 adult sheep to show nasal discharge (20%), approx. 3 having severe dyspnea (5%) and approx. another 3 having chronic intermittent diarrhea (5%). Lambs (n = 28) showed no clinical signs at all. Otherwise the flock was considered healthy and no other diseases were reported. The breeder took part in a parasite surveillance program of the Swiss small ruminant consultant association and therefore all animals had been dewormed 2–3 times per year alternating with ® ® Cydectin (Zoetis Switzerland GmbH), Endex (Novartis animal ® health, Switzerland) and Hepadex (MSD Animal Health GmbH) accompanied by an efficacy fecal parasitological monitoring. 3.2. Pathology In the post mortem examination, the mucosa of the nasal turbinates showed a bilateral symmetric, severe thickening up to
1 cm, which completely obstructed the nasal airways reaching from the nares to the naso-pharynx (Fig. 1B). The mucosa was reddish to purple, had a smooth surface and was covered with a large amount of viscous, white-grey, turbid mucus. All three animals showed a severe, diffuse, acute pulmonary edema. Histologically, the mucosa and submucosa of the nasal turbinates were severely expanded and infiltrated by mainly lymphocytes, plasma cells and less neutrophils and macrophages. Often Mott cells were present. There was severe, diffuse hyperplasia of epithelial and goblet cells (Fig. 1C). The epithelial cell layer was diffusely covered with large amounts of mucous and exfoliated cells as well as degenerated neutrophils. There were multifocal areas of necrosis and accumulations of degranulated eosinophils. Within the submucosa were multiple areas of pronounced granulation tissue. The underlying cartilage of the nasal turbinates showed moderate chondrolysis and remodeling. By immunohistochemistry, Salmonella spp. antigen was detected in large amounts within inflamed areas extraand intracellularly (Fig. 1D).
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Table 2 Results of Salmonella enterica subsp. diarizonae serotype 61:k:1,5,(7) culture of the flock of Texel sheep (n = 59) from nasal swabs and feces differentiating between age and sex. number of animals (n)
28 31 a b
sex
age
9 19 5 26
a
juvenile (lambs) juvenile (lambs)a adultb adultb
male female male (rams) female (ewes)
number of Salmonella positive samples (n) nasal swabs
feces
0 0 4 23
0 0 0 3
lambs born in spring in the same year of sampling (July 2015). >1 year old.
3.3. Bacteriology Subsequent bacteriological culture of the nasal turbinates after post mortem examination of both ewes (no. 2 and 3) revealed presence of S. IIIb 61:k:1,5,(7) (Table 1). Of the 59 investigated nasal swabs from the remaining flock, all lambs (n = 28) were negative by enrichment for S. IIIb 61:k:1,5,(7). From the 31 adults 87% (n = 27) were positive for S. IIIb 61:k:1,5,(7). From 51 fecal samples, three (6%) samples from adults were positive by enrichment for S. IIIb 61:k:1,5,(7) (Table 2). All three animals positive in the fecal samples were positive in the nasal swabs as well. The ewes in their first lactation yielded positive nasal swabs and were tested negative in the feces. All negative tested animals in the nasal swabs were also negative in the feces. 4. Discussion Three Texel sheep from a flock of 62 sheep in Switzerland either died or were euthanized due to severe illness caused by CPR in association with S. IIIb 61:k:1,5,(7). This is the first report describing CPR with isolation of S. IIIb 61:k:1,5,(7) in Switzerland, the third country worldwide, beside the USA and Spain (Meehan et al., 1992; Lacastra et al., 2012), reporting this disease. S. IIIb 61:k:1,5,(7), from the inflamed nasal mucosa, was isolated by direct bacteriological culture without enrichment, indicating a high bacterial load of Salmonella spp. Salmonella spp. antigen was co-localized within the lesions by immunohistochemistry. In previous cases in the USA, positive immuno-gold labeling for Salmonella spp. of intracellular bacilli in goblet cells within host cell membrane-bound organelles was identified by transmission electron micrograph (Meehan et al., 1992). The detection of Salmonella spp. antigen within the lesion leads to the strong assumption that S. IIIb 61:k:1,5,(7) induces CPR in sheep. In the present report, three animals with CPR were clinically ill, exhibiting chronic nasal discharge, dyspnea and respiratory distress leading to death. The examined animals suffered from these clinical signs for several months to years, which is consistent with the previously reported chronic nature of the disease (Meehan et al., 1992; Lacastra et al., 2012). Bacteriological analysis of the remaining sheep (n = 59) in the flock revealed a high nasal colonization rate with 87%of the adult sheep (n = 31) positiv. Approx. ½ (n = 15) of these colonized sheep showed clinical signs of the upper respiratory tract. Interestingly, all lambs (<6 months, n = 28) of the flock were negative while 23 of 26 of their mothers (89%) were tested positive in the nasal swabs. This shows that the nasal colonization of sheep with this specific Salmonella serovar takes several months up to a year, even if the animals are in close contact. Possible explanations are: 1) lambs are protected by maternal antibodies; 2) to become infected, animals need a time of immunosuppression/an immunosuppressive period (e.g. pregnancy, stress); 3) previous infection or co-infection by another organism (unknown) is needed. Further investigations
on weaning lambs are necessary to clarify at what time point sheep acquire colonization and infection of S. IIIb 61:k:1,5,(7). Our data leads to the assumption, that lambs can be separated from their mothers, and the remaining adult sheep of the flock, without infection during their first year in order to eradicate Salmonella in the flock. This needs further investigation, but will be an important input for authorities in case of eradication. Our findings, that lambs are not infected in the first year are supported by data of other countries, where in one case the majority of the positively tested animals were older than two years (Sanberg et al., 2002) and in another case they were older than 6 months (at slaughter) (Milnes et al., 2009). It is discussed, that S. IIIb 61:k:1,5,(7) becomes invasive if animals are stressed or immunocompromised (Greenfield and Greenway, 1973; Harp et al., 1981; Pritchard, 1990). In Norway, different stressors such as mating, vaccination and late pregnancy did not seem to influence the prevalence of S. IIIb 61:k:1,5,(7) in fecal samples (Sanberg et al., 2002). Stress may have been associated with three cases of CPR found in one of the flocks described in Spain, as the disease occurred shortly after vaccination (Lacastra et al., 2012). The most evident risk factors on herd level described in Norway were above-median herd size, having white crossbreed sheep, and higher annual replacement percentages (Alvseike and Skjerve, 2002). The outbreak of the respiratory signs and the disease of CPR in the flock started with the import of a clinically healthy ram from Germany. After introduction of the ram into the flock in 2010, several sheep of the flock apart from the ram started showing respiratory signs, suggesting S. IIIb 61:k:1,5,(7) was introduced and further transmitted by the ram to other animals in the flock. This might present a neglected way of shedding bacteria by nasal secretion or even a new path of transmission in the salmonellosis pathogenesis. The transport of the ram may have led to high stress levels and to shedding of S. IIIb 61:k:1,5,(7) leading to a transmission to other sheep in the new flock possibly by nasal discharge. Furthermore, the introduction of the new ram may have been a stressor for the remaining sheep facilitating transmission of S. IIIb 61:k:1,5,(7) from animal to animal within the flock. This is in contrast to the report by Sanberg et al. (2002) in Norway, where sharing of rams between flocks did not seem to be a risk factor for the presence of S. IIIb 61:k:1,5,(7). Furthermore, in that study, authors observed no association between prevalence and participation in ram circles; therefore, they concluded that the infection is not transmitted from animal to animal (Sanberg et al., 2002). However, the results in the present investigation strongly indicate a direct transmission from animal to animal within flocks. A representative monitoring on sheep flock is needed to evaluate the nasal prevalence of S. IIIb 61:k:1,5,(7) in both clinically healthy and CPR affected sheep. So far the epidemiology of CPR including the route of transmission and zoonotic risk analysis have not been investigated. Detailed analyses of the clinical status of sheep are mandatory to differentiate between colonization and infection.
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Through such studies, the economic impact on ovine meat and milk industry might be estimated more reliably. However, the pathogen has zoonotic potential and human infections with this distinct serovar are reported worldwide (Hall and Rowe (1992); Weiss et al. (1986); Sirmatel et al. (1998); Hjartardóttir et al., 2012). Human infections occur predominantly via the foodborne route and findings of S. IIIb 61:k:1,5,(7) in livestock are therefore of human health importance (Weiss et al., 1986; Hall and Rowe, 1992). In humans S. IIIb 61:k:1,5,(7) is among other Salmonella serotypes described as a more invasive serotype which is associated with a higher mortality rate than the low invasive serotypes (Weiss et al., 1986). Although human infections with S. IIIb 61:k:1,5,(7) rarely occur, the possible critical outcome for humans makes it necessary to pay attention and keep sheep as an important reservoir of this zoonotic organism in mind. 5. Conclusion High nasal detection rates of S. IIIb 61:k:1,5,(7) in clinically affected Texel sheep are associated with three cases of CPR. We assume that nasal colonization of S. IIIb 61:k:1,5,(7) in adults may lead to chronic nasal infections with respiratory signs and lethal mucosal proliferation in the nose. The investigation of the associated flock indicates a direct animal to animal transmission and a possible new way of shedding Salmonella by nasal discharge. Monitoring of sheep flock for nasal colonization including clinical examination is needed to understand the importance of S. IIIb 61:k:1,5,(7) in sheep production and its zoonotic risk. Furthermore, the role of carrier animals will have to be further analyzed and discussed by the official authorities. Conflicts of interest None. Funding No external funding was used for the project. Acknowledgements We thank the owner of the flock of Texel sheep Mr. Bieri, for providing us with information on the flock and giving us access to his sheep for sampling. The veterinary students Yannik Fussen and Nadine Zimmermann were very enthusiastic about sampling and we thank them for their great help with our field sampling. Further on, we thank Stefanie Müller and Isabelle Brodard for their excellent technical assistance in the bacteriological analyses, as well as Manuela Bozzo and Erika Bürgi for the histology slide preparation. Many thanks go to Dr. De las Heras, Departamento de Patologia Animal, Universidad de Zaragoza, Spain for performing the Salmonella spp. immunohistochemistry.
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