Septicemic salmonellosis in suckling piglets resulting from improper intramuscular administration of an oral vaccine

Abstract

We describe an unusual outbreak of mortality in suckling piglets following the misadministration of an oral vaccine against Salmonella Typhimurium and Salmonella Choleraesuis. Within 3–48 h of vaccination of a batch of ~700 piglets, ~300 developed marked swelling in the dorsal neck region, respiratory distress, fever, recumbency, and apathy. In total, ~100 died, and 4 were submitted for autopsy. Gross and microscopic lesions consisted of focally extensive areas of purple discoloration in the skin of the cervical region, associated with edema and hemorrhage in the subcutis and muscles. Additionally, there was interstitial pneumonia with marked interlobular edema and mild fibrinous pleuritis. Aerobic bacterial culture identified Salmonella Typhimurium (3 cases) and Salmonella Choleraesuis (1 case) in samples of skeletal muscle and lung and from pleural swab samples. Marked immunostaining against Salmonella spp. was observed in the skeletal muscle of the cervical region, as well as in blood vessels and macrophages from the lung, liver, spleen, and kidney. We concluded that inappropriate intramuscular administration of an oral vaccine against Salmonella resulted in septicemia and death in a batch of piglets.

The genus Salmonella is composed of gram-negative, rod-shaped, facultatively anaerobic bacteria that are divided into 2 species: Salmonella enterica and S. bongoni. ⁷ S. enterica is most frequently associated with clinical disease in domestic animals, with > 2,600 serovars identified.^7,8 In pigs, 2 main serovars of S. enterica are linked to clinical disease: Salmonella Choleraesuis, typically causing septicemic clinical disease; Salmonella Typhimurium, generally causing enteric clinical disease. ⁷ Infection occurs mainly through fecal-oral transmission, and most Salmonella infections are subclinical. ⁴

Salmonellosis can affect pigs of all ages, but it is observed most commonly in nursery and growing-finishing pigs.^2,7 The disease rarely occurs at an early age, presumably because of the transfer of maternal immunoglobulins.^{6 –8} However, a study in Brazil found a 6.15% frequency of salmonellosis in suckling piglets with septicemic and enteric clinicopathologic presentations. ⁸ Preweaning salmonellosis may be linked to environmental contamination, inadequate cleaning and disinfection of facilities, management mistakes, introduction of carrier animals, and poor infrastructure.^1,7,8 Furthermore, implementing effective measures to control and prevent salmonellosis can be challenging. These measures include biosecurity practices, adequate cleaning and disinfection, adopting all-in, all-out management, administration of antimicrobials in clinical cases, and the use of vaccines.^7,12

Vaccination is a widespread practice in commercial swine production and is the most efficient and cost-effective method for controlling infectious diseases. ¹⁰ However, the success of vaccination relies on several factors, involving the swine, the vaccine, and the vaccination protocol. ⁵ Some vaccination failures can result in mortality and may be attributed to improper storage of vaccines, needle size and gauge, dosage errors, inappropriate route of vaccine administration, use of expired vaccines, and the timing of vaccination.^3,5,10 We describe here an unusual outbreak of mortality in suckling piglets that resulted from the misadministration of an oral vaccine against Salmonella Typhimurium and Salmonella Choleraesuis.

In a commercial pig farm, the farm workers were instructed by the veterinarian to implement a new vaccination protocol against Glaesserella parasuis (IM application in the neck, commercial vaccine, 2 mL/pig), Streptococcus suis (IM application in the neck, autogenous vaccine, 2 mL/pig), and Salmonella Typhimurium and Salmonella Choleraesuis (oral application, live-attenuated commercial vaccine, 2 mL/pig). Approximately 700 piglets 7–13-d-old were vaccinated at the same time. However, 3 h after vaccination, some animals developed nonspecific clinical signs, including apathy, acute respiratory distress, and recumbency. Furthermore, the piglets also had fever and marked swelling of the dorsal neck region, which raised suspicion that bacterial contamination had occurred at the injection site during vaccination. Another suspected diagnosis was the misadministration of the oral vaccine, although this information was not provided by the pig farm workers to the veterinarian in charge of the farm. As a result, an antibiotic was administered to all ill piglets in an attempt at treatment (IM enrofloxacin in the neck, 7.5 mg/kg, Baytril; Bayer). Clinical improvement was noted, such as decreased swelling in the cervical region and in fever, improvement of respiratory signs, and increased suckling; however, ~300 piglets had clinical signs, and ~100 died within 48 h of the vaccination; 4 unmedicated piglets were sent for autopsy to confirm the diagnostic hypothesis.

In the 4 autopsied piglets, we observed focally extensive areas of purple discoloration extending from the dorsal to the ventral cervical region of the skin (Fig. 1), associated with marked edema and hemorrhage in the adjacent subcutaneous and skeletal muscle tissues. Additionally, the lungs were non-collapsed with pronounced interlobular edema and mild deposition of fibrin on the pleural surface (Fig. 2); 3 of the 4 piglets had moderate splenomegaly. No significant pathologic changes were observed in the remaining tissues. Samples of lung, trachea, heart, esophagus, lymph nodes, liver, spleen, kidneys, small and large intestines, brain, spinal cord, urinary bladder, gall bladder, skeletal muscle, and skin were collected, fixed in 10% neutral-buffered formalin, and processed routinely for histologic examination. Samples of lungs, skin, and subcutaneous tissue (cervical region), as well as pleural swabs, were collected during autopsy and submitted for bacteriologic analysis.

Figures 1, 2.

Gross findings of septicemic salmonellosis in suckling piglets resulting from improper administration of an oral Salmonella vaccine. Figure 1. Four piglets with focally extensive purple discoloration in the skin of the ventral cervical region. Figure 2. The lungs are non-collapsed with pronounced interlobular edema and mild deposition of fibrin on the pleura.

Histologically, the dermis, subcutaneous tissue, and adjacent skeletal musculature of the cervical region of all 4 piglets were markedly distended by edema and hemorrhage (Fig. 3), and associated with mild necrosis and mild infiltrates of neutrophils (Fig. 4). Moreover, mainly among skeletal muscle fibers and in the subcutaneous tissue, rod-shaped bacteria were observed. Interstitial pneumonia was present, composed of mild infiltrates of lymphocytes, macrophages, and neutrophils in alveolar septa, associated with marked alveolar and interlobular edema, and mild multifocal fibrinous pleuritis. Furthermore, there was congestion of the spleen and microthrombosis of capillaries of glomerular tufts, with aggregates of bacteria present. No significant microscopic lesions were observed in other tissues. S. enterica subsp. enterica grew in culture from 4 of 4 lung samples, serotyped as Salmonella Typhimurium (3 of 4) and Salmonella Choleraesuis (1 of 4; Table 1). Bacterial isolation under anaerobic conditions was negative.

Figures 3–6.

Microscopic findings of septicemic salmonellosis in suckling piglets resulting from improper administration of an oral Salmonella vaccine. Figure 3. Subcutaneous tissue from the cervical region with marked edema and hemorrhage. H&E. Figure 4. Skeletal muscle from the neck with a mild inflammatory infiltrate of neutrophils and moderate edema. H&E. Figure 5. Marked immunolabeling for Salmonella spp. between myocytes from Figure 4. Immunohistochemistry (IHC). Figure 6. Immunolabeling for Salmonella spp. in renal glomeruli. IHC.

Table 1.

Bacteriological, serotyping, and immunohistochemical results of salmonellosis in piglets resulting from improper route of administration of an oral vaccine against Salmonella Typhimurium and Salmonella Choleraesuis.

Piglet	Bacterial isolation			Salmonella serotype	Immunohistochemistry (Salmonella spp.)
	Skin*	Lung	Pleural swab	Lung	Skin*	Lung	Spleen	Liver	Kidney
1	+	+	–	Choleraesuis	+	+	+	+	+
2	–	+	–	Typhimurium	+	+	+	+	–
3	–	+	+	Typhimurium	+	+	–	–	–
4	+	+	+	Typhimurium	+	+	+	+	+

^*Subcutaneous tissue and skeletal muscle (cervical region).

Samples of skin, lung, spleen, kidneys, and liver from all pigs underwent immunohistochemistry (IHC) analysis for Salmonella spp. Endogenous peroxidase activity was inhibited by immersing the tissue sections in a 10% hydrogen peroxide–methanol solution for 10 min. Antigen retrieval was performed enzymatically using 0.05% protease XIV for 15 min at 37°C. The slides were incubated overnight at 4°C with polyclonal anti-Salmonella antibody (1:1,000, Salmonella 8209-4006 rabbit anti-Ig; Bio-Rad). The Romulin AEC chromogen kit (Biocare Medical) was used, with Harris hematoxylin counterstain. Positive IHC controls were formalin-fixed, paraffin-embedded intestinal samples from a pig with enteric salmonellosis confirmed by bacterial culture. As a negative control, the primary antibody was replaced with rabbit antiserum (Rabbit IgG, universal negative control serum; Biocare Medical).

IHC results revealed marked immunostaining for Salmonella spp. within the subcutaneous tissue and skeletal muscle of the cervical region (Fig. 5), in the cytoplasm of macrophages, and in blood vessels from the lung and spleen, Kupffer cells in the liver, and renal glomeruli (Fig. 6).

Based on clinical signs and gross and microscopic lesions, along with the presence of Salmonella spp. in the skeletal muscle and subcutaneous tissue of the cervical region of all piglets (confirmed by IHC), we concluded that there was inappropriate IM administration of an oral Salmonella vaccine. The administration of a vaccine via the wrong route can trigger the disease, even though the inoculum contained live-attenuated Salmonella. We believe that inoculation of Salmonella triggered septicemia and death as a result of septic shock, with lesions of interstitial pneumonia, fibrinous pleuritis, and glomerular microthrombosis.

Bacterial contamination of injection sites or skin wounds has been documented in pigs, mainly by Clostridium spp., resulting in edema, hemorrhage, local myonecrosis, and, in several cases, death by septic shock. ¹⁴ In these histotoxic gas-gangrene infections caused by Clostridium spp., the affected tissues are also swollen and warm, accompanied by red-purple skin discoloration and crepitation. Such infections can be fatal in < 24 h.^11,14 Some of the lesions present in gas gangrene are similar to our findings and represent an important differential diagnosis; however, we promptly ruled out this diagnosis based on the results of histopathologic findings and bacterial isolation and IHC of Salmonella of the lesions.

Other important differential diagnoses, given the systemic lesions and clinical signs observed, are septicemic diseases that may occur in young pigs, such as colibacillosis, actinobacillosis, and salmonellosis (in cases of fecal-oral infection). These conditions may potentially result in sudden death and high morbidity in suckling pigs. Specifically, salmonellosis might have been misdiagnosed if the skin lesions had not been carefully analyzed in relation to the clinical history. Additionally, the septicemic form of salmonellosis may not have obvious intestinal lesions ⁷ ; we did not observe intestinal alterations in any of the submitted pigs. This emphasizes the importance of bacterial isolation and IHC of skin lesions (skeletal muscle and subcutaneous tissue) to confirm the etiology involved and to corroborate the hypothesis of an inappropriate route of vaccine administration.

Although the clinical and pathologic features of Salmonella infections may vary, gross and microscopic findings are well described in swine. ⁷ Briefly, in the septicemic form, gross lesions include marked interlobular pulmonary edema, splenomegaly, enlarged lymph nodes, 1–2-mm foci of hepatic necrosis, and reddened intestines. Histologically, foci of coagulative necrosis in the liver and spleen, diffuse histiocytic interstitial pneumonia or suppurative bronchopneumonia, and microthrombi in venules of various tissues may be present.^7,9,13 We observed most of these pathologic findings in our cases (interstitial pneumonia, pleuritis, splenomegaly, renal microthrombosis), which were confirmed through bacterial isolation and/or IHC. The lesions that we observed were less pronounced, possibly given the short clinical evolution, early death of the animals, and atypical inoculation of the Salmonella vaccine. Correct oral administration of this live-attenuated vaccine should not have caused mortality.

Septicemia caused by Salmonella spp. is usually associated with a host-adapted serovar (i.e., Salmonella enterica subsp. enterica serovar Choleraesuis in pigs).^4,13 Nevertheless, infections with other S. enterica serovars, such as Salmonella Typhimurium and Salmonella Anatum, may also result in systemic lesions in piglets, ⁷ as we found in our cases of Salmonella Typhimurium septicemia.

Although immunization, particularly against Salmonella Choleraesuis, is not a routine practice in many commercial pig farms in Brazil, it is worth emphasizing that, historically, the reduction of septicemic salmonellosis outbreaks has been attributed to improvements in management practices on farms and the use of live-attenuated vaccines.^4,7 Vaccination against Salmonella is a challenge, and the efficiency of the protocol is linked to several factors, including adequate management, serovar, age, and stage of production involved, competing diseases, and type of vaccine.^4,15 Commercial vaccines are licensed, tested, and approved in experimental studies, including the appropriate route of administration. ¹⁵ However, the success of vaccination includes the correct route of application. Pig farm management must always be monitored or carefully guided by a veterinarian.

When new vaccine protocols are implemented, especially when significant changes are made in vaccine handling and administration route, it is crucial to provide training to farm teams and collaborators to prevent fatal errors.