1. Introduction
Salmonella enterica subsp.
enterica infections pose a threat to the dairy cattle industry due to economic losses in infected herds, mainly because of the costs of clinical disease, which include diagnostic laboratory investigations and treatment of clinical cases. Cleaning and disinfection, as well as control and prevention measures, pose additional costs [
1].
Salmonella Dublin,
S. Typhimurium, and the
S. Typhimurium monophasic variant 1,4,[5],12:i:- are generally associated with salmonellosis in calves and adult cows, causing mild to severe illness [
1]. In addition, other, less frequently detected serotypes must be considered relevant to cattle farms. Clinical signs of bovine salmonellosis may include diarrhea, fever, anorexia, and dehydration, while in milking animals, milk production severely drops and abortions may also occur [
2]. Infected animals can become shedders and spread the organism for a varying period of time, as well as intermittently in the environment, after either clinical or subclinical infections. In favorable conditions,
Salmonella spp. can survive in the environment outside the host for a long time [
3,
4]. Transmission between animals is fecal–oral, and the contamination of the environment, feed, and water play an important role in the epidemiology of salmonellosis [
3]. Herd management is a crucial factor; free stalls compared with tie-stall housing systems have been described to increase contact between animals, and in larger herds, cows and calves may be more densely housed, which could promote the spread of infections and increase environmental contamination. Some European countries have implemented national or regional surveillance, control, or eradication programs [
5], while in Italy, there is no national control program for salmonellosis in cattle. Thus, data on the occurrence of
Salmonella spp. in cows are collected in the framework of Directive 2003/99/EC [
6]. Nevertheless, clinical salmonellosis is a notifiable disease in Italy. Considering the increasing importance of salmonellosis in cattle and its potential impact not only on animals but also on public health, it is of pivotal importance to optimize the data collection system, as well as to standardize the methods used for epidemiological investigations in case of outbreaks [
7].
These considerations led to the need to find strategies to manage Salmonella outbreaks. A research project funded by the Istituto Zooprofilattico Sperimen tale della Lombardia e dell’Emilia-Romagna (IZSLER), AUTOFIN_SALMONEL_BOV “Drafting a manual for the management of salmonellosis outbreaks in dairy farms”, focused on managing Salmonella outbreaks on dairy farms in the Emilia-Romagna region. The present study describes an outbreak of S. Bredeney in a dairy herd and the consequent control strategies applied.
2. Materials and Methods
2.1. Farm Characteristics
This case report involved an intensive, loose-housing dairy farm for the production of Parmigiano-Reggiano cheese with a total of 600 heads of Holstein Friesian cattle, with a year-round calving pattern. Milking cows (300) had an average daily production of 34 kg of milk/cow. The average productive lifespan of the cows was 2.4 lactations. The most frequent reasons for culling were aging, mastitis, and lameness, with an average of 35% cows culled. Artificial insemination procedures were performed. The resting area type for lactating cows consisted of cubicles with straw bedding materials. Dry cows also had an external paddock.
Colostrum was stored in a colostrum bank and valued by using a Brix refractometer. On this farm, calves were usually fed 4 L of colostrum only once after birth.
For the first two months of life, calves were housed in single boxes, while older calves were housed in boxes with straw bedding material with other calves of the same age. After collection and storage, slurry was deposited on pastures. No animals had been purchased in the last few years. Barns were naturally ventilated, while milking cows’ barns were ventilated by fans. In addition, there were misting fans in the milking parlor and waiting area. Natural light was sufficient during the day, and lighting control was available during the night, combined with an 8 h dark period. The farm used 60% home-grown hay and grass for their livestock, without using silage or fermented forage. Drinking well water was provided in a livestock water trough and was analyzed once a year. Straw bedding materials were changed once a month, manure scrapers were used twice a day, and straw bedding was added to calves’ cages every other day and cleaned using high pressure washers when calves were moved to the post-weaning youngstock boxes. Rodent and insect control measures were performed, while there were no control measures for wild birds or synanthropic animals. Antimicrobial treatments data showed that antibiotic use for the year 2020 was 6.32 Defined Daily Dose Animal for Italy (DDAit), higher than the national median (2.56 DDAit).
The vaccination schedule included a live attenuated intranasal bovine parainfluenza 3 virus (PI3) and bovine respiratory syncytial virus (BRSV) vaccine in calves; inactivated vaccine for bovine coronavirus (BCoV), bovine rotavirus (BRV), and Escherichia coli administered intramuscularly in dry cows; infectious bovine rhinotracheitis (IBR) marker vaccine in milking cows; and inactivated bovine viral diarrhea virus (BVDV), inactivated PI3 vaccine, and live BVRS vaccine in milking cows.
2.2. Outbreak Description
In July 2021, two deceased 7-day-old calves were sent to the Diagnostic Laboratory of Parma (IZSLER) for post-mortem examination and laboratory investigations. The practitioner reported that the calves initially showed watery diarrhea and hematochezia, followed by weakness, recumbency, severe dehydration, and death. No enteric disorders, abortions, or other significant clinical signs were observed in cows. Necropsy, performed on both calves using standardized procedures, showed severe dehydration, sunken eyes, and yellowish, blood-stained, foul-smelling scour around the perineal region and tail. Gross lesions observed were hepatomegaly, enlarged spleen, catarrhal to catarrhal–hemorrhagic enteritis, catarrhal abomasitis, mesenteric lymphadenitis, and pulmonary hemorrhages. Bacteriology was performed from the liver, kidney, spleen, intestinal content, lymph nodes, and lungs of each calf. Briefly, samples from the tissues reported above were plated on blood agar and on Gassner agar (a selective medium for
Enterobatteriaceae). The latter allows for the differentiation of lactose-fermenting (such as
E. coli) and lactose-non-fermenting Gram-negative enteric bacilli. Plates were incubated aerobically overnight at 37 °C. Subsequently, lactose-negative colonies were further plated on Xylose Lysine Deoxycholate agar (XLD) (Oxoid Holding Ltd., Hants, UK) and Brilliant Green Agar (BGA) (Biolife S.R.L., Milano, Italy) and incubated overnight at 37 °C [
8]. Colonies with morphology characteristic of
Salmonella spp. were typed with biochemical and serological methods. Serotyping by serological rapid agglutination techniques, through the characterization of somatic, flagellar and capsular antigens, was performed according to ISO/TR 6579-3:2014 [
8,
9,
10]. Final serological characterization was performed according to the scheme of Kauffmann–White–Le Minor [
11]. Bacteriological investigations led to the isolation of
S. Bredeney from all samples collected from both calves.
Antimicrobial susceptibility testing was performed on the strain isolated from tissue samples collected during the necropsies, by the Kirby–Bauer disk diffusion method, using a panel of 10 antimicrobials: nalidixic acid (30 µg), amoxicillin and clavulanic acid (20–10 µg), ampicillin (10 µg), cefazolin (30 µg), enrofloxacin (5 µg), florfenicol (30 µg), gentamicin (10 µg), kanamycin (30 µg), tetracycline (30 µg), and trimethoprim + sulfamethoxazole (1.25–23.75 µg). The
Salmonella spp. isolate was classified as susceptible, intermediate, and resistant, following CLSI interpretative criteria [
12]. The
Salmonella Bredeney strain tested was resistant to kanamycin and tetracycline, but it was susceptible to all the other antimicrobials tested.
Investigations for BCoV [
13], BRV [
13], and BVDV [
14] were performed by PCR. The samples were positive for bovine coronavirus, while BRV and BVDV were negative.
2.3. Outbreak Management
Outbreak management consisted of four main steps. In the first step, a farm visit was conducted in order to perform an epidemiological investigation and to assess the welfare and biosecurity levels of the herd, the prevalence of
S. Bredeney, and the presence of other
Salmonella serotypes. The epidemiological investigation, as well as welfare and biosecurity levels, were assessed using a checklist for the control of
paratuberculosis and
Salmonella Dublin [
15], and a risk scoring form and graphics were used to visualize the high-risk areas in the herd and to evaluate transmission pathways. These spread-sheets are appendices to a Manual for Advisors in Denmark. The risk scoring form was used to go through the herd systematically and assign risk scores to different relevant barn sections and management practices. The maximum scores were decided by the authors of the manual according to existing knowledge about risk factors for the spread of
S. Dublin in cattle herds, and they were weighted so that the most critical areas for control counted most in the total sum of risk scores [
16,
17].
The prevalence of
Salmonella spp. was assessed by collecting rectal swabs from all calves (from birth to weaning age), boot swabs from youngstock’s, dry cows’, and milking cows’ boxes, and sponge sticks on manure scrapers (
Table 1). All these samples were tested using bacteriological techniques that included an enrichment step [
8], and bulk milk samples were tested using real-time PCR [
18].
In the second step, a health management plan (HMP) was drafted to summarize and evaluate the risk scores and to plan actions based on the results of the epidemiological investigations carried out and analytical results obtained in step 1. The HMP was made up of a set of measures that consider the characteristics of the farm, such as its structural and hygienic elements. The HMP, implemented and shared with both the farmer and the practitioner, was a tool to increase the hygiene of livestock farming and to improve its health and welfare aspects with direct and indirect prophylaxis measures, aiming to eliminate or to strongly reduce salmonellosis cases.
In the third step, the measures contained in the HMP were applied, which first consisted of a deep cleaning protocol.
The fourth step was characterized by a monitoring period, during which individual samples were collected by the public health authority from calves that showed clinical signs, from cows 3 weeks before parturition, and from all 2-day-old calves to detect infected animals and to rule out chronically infected animals. Six months after the end of the monitoring period, a follow-up was performed by a farm visit repeating the same environmental sampling performed during the first farm visit and reported in
Table 1 (calving cow area, dry cow area, and youngstock area) and to re-evaluate management practices with the checklist previously used.
3. Results
3.1. First Step
After the farm visit, the laboratory investigations showed that 48% of rectal swabs collected from calves were positive for
S. Bredeney, and only six of them belonged to diarrheic calves, all of them under 14 days of age. Five out of fifteen (33%) environmental samples collected from the milking cows’ group, dry cows’ group, and youngstock with sponges on automatic passageway scrapings, boot swabs, and pooled samples of feces were positive for
S. Bredeney. Bulk tank milk analysis was negative (
Table 2).
The epidemiological investigation on the first visit highlighted scarce biosecurity levels in the herd. In particular, external biosecurity analysis showed that the main critical aspects revealed by the checklist were the absence of a vehicle disinfection system at the entrance to the herd and the rendering service for the final removal of carcasses, which usually passed through the herd, near the cows’ housing areas. Internal biosecurity evaluation showed that the all-in all-out principle was not respected, overcrowding was observed in the milking cows’ and dry cows’ area, bedding material removal and replacement was on a monthly basis, the same boots and clothes were used in different barns, and no quarantine box was available for every category of animals; also, synanthropic animals such as cats, pigeons, starlings, and hens were present in the herd. In addition, there was an external paddock for dry cows, so contact with wild animals was possible.
Figure 1 and
Figure 2 show the risk score graphics from the epidemiological investigation checklist. The first figure reports a risk score graphic for the pre-weaning calves’ area, as pre-weaned calves were the main category involved in the outbreak; the second figure shows the total risk score for the entire area of the herd and possible risks from other herds (
Figure 1 and
Figure 2).
3.2. Second and Third Steps
After the first visit, the HMP was drafted as reported above in order to find strategies to reduce the prevalence of S. Bredeney in the herd and also to suggest ways to improve the biosecurity levels and the hygiene parameters, in particular on passageways scrapers and housing areas, to reduce the contamination of dried fecal material. The cleaning protocol was planned to include descaling and removal of all organic material from the structures, in particular the calves’ pens, calving maternity pen, and manure scrapers in the milking cows’ and dry cows’ area. All structures, means of transport used for livestock, and tools used in livestock farming suspected to be contaminated had to be adequately cleaned, so it was suggested that movable tools, wood, ropes, and all organic material be removed before applying the disinfectant.
The suggested cleaning protocol consisted in the following procedure: scraping and removing coarse dirt mechanically (with the aid of brooms, vacuum cleaners, blowers, etc.); initial rinsing with hot water at a temperature higher than 45 °C, but lower than 60 °C, to dissolve fats and facilitate their detachment; then, applying the detergent in order to loosen dirt from the surfaces and allow it to move away with the next rinse. Subsequently, the protocol included rinsing with water at tap temperature, then applying the disinfectant after drying the surfaces, according to the dilution and prescribed methods of use; leaving the disinfectant to act for at least 6 h; and finally, rinsing with water at tap temperature, preparing bedding on dry surfaces and reintroducing the animals. The same treatment was suggested for feeders, drinkers, calf milk buckets, teats for calves, cages, etc.
The cleaning protocol also involved a workflow which suggested that operators of the calving area began cleaning procedures with healthy animals and subsequently moved on to infected animals. High-pressure washing was not recommended due to the risk of environmental cross-contamination and the aerosolization of contaminated material, which can increase the risk of infection in workers and animals. High-pressure washers effectively remove coarse material, such as manure, but they are not effective in eliminating bacterial biofilms. A list of effective disinfectants was also suggested. The disinfectant used on this farm was quaternary salts of ammonium because they are effective on Gram-negative bacteria such as Salmonella spp. and were already available on the farm.
It was also recommended to keep positive animals separated from the others, so it was important to define an area as a hospital pen. The use of disposable boots and gloves for different areas was prescribed. Disposable gloves were used in the calves’ pen during daily cleaning procedures and a boot sanitization point was implemented at the entrance to the calving area, because the use of dedicated boots or use of disposable shoes/overshoes was not feasible for operators.
The entrance of synanthropic and wild animals was forbidden. In addition, corrective measures were adopted, such as the improvement of routine hygiene procedures, implementation of an appropriate pest control system, systematic cleaning and disinfection of new-born calves’ cages, and proper disinfection of the equipment used to feed calves (
Table 3).
Male calves were usually sold to other finishing farms, so to control the spread of infection to veal calf farms, all new-born calves were sampled at least twice within 7 days, and the movement of positive calves was forbidden. According to the HMP, positive clinically affected animals were isolated and treated with antimicrobials. Animal movement or reintroduction was scheduled only after at least two negative bacteriological tests on rectal swabs, the first carried out 3 days after the end of treatments, the second 5–7 days after the first negative sample. In case of repeated positive results, it was advised that animals be culled. It was suggested that asymptomatic animals be isolated and avoid antimicrobial treatment. Animal movement was allowed only after at least two negative tests, as mentioned above [
19].
In Italy, there are no commercial vaccines available for Salmonella spp. in cattle, so in cases where vaccination is considered to be a control tool, a Salmonella-inactivated autogenous vaccine would be necessary. However, in this case, vaccination was not adopted during this outbreak, as the farmer, in agreement with the practitioner, was not willing to add a vaccination to the vaccination plan.
3.3. Fourth Step
During the monitoring period, from September to November 2021, 21% of rectal swabs collected from calves were positive for
S. Bredeney (
Figure 3 and
Figure 4).
After two negative results were obtained on all samples collected at the end of October and at the beginning of November 2021, the monitoring period was interrupted. All the environmental samples collected six months after the end of the monitoring period (follow-up visit) were negative and new cases of salmonellosis were not observed.
Figure 5 and
Figure 6 show that risk scores were reduced in comparison with the first visit, especially for pre-weaning calves. The total score for calves after weaning and heifers remained the same because the structural risk elements of the herd could not be improved. The results obtained, together with the absence of clinical signs of salmonellosis and the adoption of the HMP by the practitioner and farmer, led to the revocation of further control measures.
4. Discussion
Salmonellosis is a significant disease in animals, both due to the possible health and economic impact and due to the public health implications resulting from the zoonotic nature of the infection [
20,
21]. Possible forms of
Salmonella spp. infections are enteric, septicemic, and reproductive. Although reproductive losses are only of concern in adult cows, enteric disease can be seen at any age, from newborn calves through to adulthood [
22,
23].
Bovine salmonellosis can be caused by different serotypes; however, the most frequent, responsible for the majority of reported cases, are
S. Typhimurium, including its monophasic variant
S. 1,4,[5],12:i:-, and
S. Dublin, a host-adapted serotype [
24,
25].
In fact, as reported by the 2018 Entervet report [
26], the most frequently isolated serotypes in cattle are
S. Typhimurium and its monophasic variant, identified in 42.6% of the total isolates from cattle in 2018, followed by
S. Dublin, with an incidence of 31.8%.
A retrospective study of
S. enterica strains submitted to the Wisconsin Veterinary Diagnostic Laboratory from 2006 to 2015 showed that among 5000 isolates detected,
S. Dublin was the most prevalent serotype identified, accounting for a total of 1153 isolates (23% of the total) [
27]. Along with
S. Dublin,
S. Cerro (16%),
S. Newport (14%),
S. Montevideo (8%),
S. Kentucky (8%), and
S. Typhimurium (4%) comprised the top six most isolated serotypes. In a comprehensive study from the north-eastern United States in 2009, more than 800 dairy herds were involved. The most frequently detected serotype was
S. Newport, accounting for 41% of cases, followed by
S. Typhimurium, accounting for almost 20% of cases [
28]. Even though
S. Bredeney is not frequently detected in cattle, it was relevant in this case. In fact,
S. Bredeney is sporadically identified in cattle but, as in this case, can represent a relevant serotype, able to bring about disease and an important economic impact [
29]. There are few publications regarding
S. Bredeney in cattle, but in 1994, Marley et al. [
30] reported that
S. Bredeney infections in cows had frequently been reported in France. These authors described an outbreak on a farm comprising 160 milking cows. During the first 2 months of the outbreak, cases of fever (68% of cows), dysentery (80%), and abortions (6.9%) were associated with
Salmonella isolations in feces or in products of abortion.
S. Bredeney was recovered from the milk tank, but individual milk sampling was not practicable. Authors report that in that specific outbreak, in several respects, the results were similar to those observed with
S. Dublin, a serotype considered to be primarily adapted to cattle. Cormincan et al. [
29] reported that
S. Bredeney is a well-recognized serotype isolated from poultry, other animals, and the environment, and that it is an uncommon human pathogen associated with occasional outbreaks. The authors also added that
S. Bredeney had emerged as the third most identified serotype among human clinical isolates, with reference to the Irish National Salmonella Reference Laboratory in the years 1998 to 2000. In 1998, Baker and colleagues [
31] reported that
S. Bredeney had been frequently isolated from a range of food products, animal sources, water, and sewage effluent in the period 1988–1993.
In the outbreak reported in this study, bulk tank milk analysis was negative, and sampling was conducted by the local public health authority due to the destination of the milk produced and the possible associated risks. In this regard, it is important to highlight that in the production of long-seasoned cheeses (Grana Padano and Parmigiano-Reggiano), the high cooking temperatures (55 °C), salting, acidification of the curd carried out by lactic bacteria, and long seasoning lead to the complete inactivation of
Salmonella spp. [
32]. Therefore, the risk of salmonellosis linked to the consumption of this type of dairy products appears negligible [
32].
In the outbreak described in the present study, different areas of the farm and a high number of samples from calves tested positive, which made huge impact on the herd. Also, the management of the outbreak lasted a few months.
The most important risk factors observed on the farm were the frequency of cleaning, the presence of synanthropic animals, and the misuse of protective clothing. Overcrowding and poor environmental hygiene, as observed in both milking cows’ and dry cows’ areas, were considered predisposing factors for the spread of the infection. Improving cleaning and disinfection procedures during the outbreak and during the monitoring period was considered a key point to reduce the spread of
Salmonella within the herd. A specific protocol of workflow and hygiene, as suggested in the HMP, should be considered an important measure to reduce the incidence of clinical cases, especially in the calving area. In this study, indirect prophylaxis, thereby implementing internal biosecurity, can be considered crucial to gradually reducing cases of salmonellosis on the farm. In most cases, there are multiple factors involved in precipitating the
Salmonella outbreak and in order to be successful, health management plans have to be designed based on specific characteristic of the farm, as considering different aspects of the outbreak and the serotype involved are of critical relevance. For instance, the host-adapted serotype
S. Dublin has the specific ability to produce long term carriers that shed bacteria in the environment, therefore becoming a steady source of infection [
33,
34]. In our study, the serotype involved was
S. Bredeney, a non-host-adapted serotype that is rarely detected in cattle; therefore, our health management plan focused more on increasing levels of biosecurity and disinfection practices.
The activities described in this case report, such as the production of an HMP and the strategic measures contained therein, allowed for the definition of targeted and joint actions with the involvement of different professional figures such as public health veterinarians, the practitioner, and the farmers. The actions undertaken made it possible to obtain an improvement in some critical points present on the farm and to contribute to the management of the salmonellosis outbreak described.