Management of zoonoses in research institutions – lessons learned from a Coxiella burnetii outbreak case

Animal health monitoring

The preliminary animal examinations at the farm of origin one week before and the clinical examination upon delivery were unremarkable and fulfilled all requirements for placing the animals in the facility. A group of 12 sheep were transported to the facility between the 129th and 134th day of pregnancy in accordance with Regulation (EC) 1/2005. According to daily obstetrical examinations, the lambs were born between the eighth and the 17th day after arrival. The ewes were transported back to the farm of origin 14 days after delivery, except one ewe showing reduced general condition and increased recumbency over the next two weeks, which therefore remained at the facility for 10 more days under veterinary supervision until recovery allowed transport.

Contact examination

Nineteen days after the animals’ arrival, respectively 11 days after the birth of the first lamb, the first individuals from the group directly involved in the experiment (scientists, animal caretakers, veterinarians) developed flu-like symptoms, such as mild headache, fatigue and malaise, up to several days of high fever and other clinical symptoms. Three people required hospitalization for one to several days.

Because of the clustering of cases with similar symptoms a presumptive diagnosis of Q fever was made seven days after the first case. The blood sera of the contacts were tested by enzyme-linked immunosorbent assay (ELISA) and immunofluorescence technique in the Q fever conciliar laboratory (Landesgesundheitsamt Baden-Württemberg (LGA), Stuttgart, Germany) (Supplementary material supplement 7 online).

Based on facility access documentation, a total of 110 possible contact persons were identified and made an offer to participate in an occupational health screening performed by a company doctor according to §5(2) of the Ordinance on Preventive Occupational Health Care (ArbmedVV, Germany ²⁷ ). Persons with possible direct contact with the animals or premises where the animals were kept were prioritized and serologically tested first, followed by all others.

Monitoring of titres was offered and carried out by the Occupational Medicine Centre in accordance with the recommendations of LGA. Those recommendations were based on the initial titre results. If the initial tests resulted in positive titres, the borderline cases were retested some weeks later, but most people were retested three and six months after the incident to rule out residual titres or a chronic course of the disease.

Examination of the environmental samples from the rooms

The facility’s large animal holding rooms were subject to annual routine examination for Cb by a polymerase chain reaction (PCR ²⁸ ) (Supplement 5) method from environmental dust samples prior to the infection event. According to a standardized scheme (Supplement 1) the rooms were swabbed and tested by the reference laboratory of Lower Saxony State Office for Consumer Protection and Food Safety (LAVES, Hannover, Germany).

Environmental dust samples of the first sampling (Supplement 1) were collected from animal rooms and analysed by LAVES (PCR ²⁸ ) (Supplement 5) (Figure 1).

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Figure 1.

Process in the present case in the form of a flow diagram.

To confirm the initial suspicion, an additional litter sample collection (Supplement 2) was examined.

For the second sampling (Supplement 1) in the first review phase (Figure 1), the animal housing corridors walls were equally swabbed.

In a third sampling (Figure 1), all other rooms of the facility (small animal husbandry, storage, office rooms) were sampled (Supplement 1).

According to the requirements of the competent authority, State Office for Occupational Safety, Health Protection and Technical Safety (Landesamt für Gesundheit und technische Sicherheit (LaGetSi)), all disinfected rooms had to test negative twice by PCR to prove the effectiveness of the procedures used and to obtain official approval for the resumption of operations.

Personal protective equipment (PPE) under enhanced safety measures

In addition to the standard PPE (dedicated clothing and shoes for the area, gloves, FFP2 mask), a disposable protective overall (Tyvek®-500Xpert-Overall, DS SafetyWear GmbH, Lohmar, Germany) with battery-powered respiratory filter device and head hood (SR500, SR 561, Sundström Safety AB, Lagan, Sweden) with a combined particle/chemical filter was used. Sampling after the disinfection was performed using standard PPE.

Examination of large animals (sheep, pigs)

The relevant ewe was identified during the initial sampling (Figure 1) and examined for Cb by PCR detection ²⁸ (LAVES) (Supplement 5) using a vaginal swab.

In the third sampling (Figure 1), samples were taken from remaining separated pigs by fur swabs in the spinal region of the animals and from sheep by vaginal swab.

Examination of small animals (mice, rats, guinea pigs, rabbits)

After positive findings of the second sampling (Figure 1), the entire small animal population was also examined for further risk assessment via collective fur swabs (spinal region) of the animals and dust swab samples from cages, exhaust air ducts of the individually ventilated cage (IVC) housing systems and the airflow (LAF) housing cabinets.

Blood samples from existing sentinel animals (mice, rats) were sent to FLI (Nationales Referenzlabor Friedrich-Löffler-Institut, Jena, Germany) for testing for Q fever antibodies by means of a modified ELISA ²⁹ (Supplement 4) and, after painless killing of the animals, also spleen samples were sent to FLI for PCR detection (Supplement 6). In parallel, pooled spleen, lung and liver samples from the sentinel animals were sent to LAVES for PCR examination ²⁸ (Supplement 5).

Investigation of the ventilation and air conditioning system of animal husbandry

To exclude pathogen spreading through the air conditioning system into the environment, dust swab samples were taken (Supplement 3) for PCR detection ²⁸ (LAVES) (Supplement 5).

Carrying out the cleaning, disinfection and decontamination work

The entire facility was cleaned and disinfected in several steps and under increased safety precautions (see section on PPE above).

Cleaning of livestock rooms

Animal bedding and excrement were collected as infectious waste and disposed of separately. Animal rooms were mechanically cleaned using a high-pressure cleaner (Hochdruckreiniger, Alfred Kärcher Vertriebs-GmbH, Winnenden, Germany) and then surface-disinfected using a foam disinfection device (WOFA-MAT®, Kesla Hygiene AG, Bitterfeld-Wolfen, Germany). A German Veterinary Society-listed antimicrobial disinfectant with the active ingredients peracetic acid with acetyl hydroperoxide, hydrogen peroxide and acetic acid (Wofasteril® 2%ig, Kesla Hygiene AG) was used.

Wipe disinfection

Outside the animal holding rooms, all ancillary, operating and office rooms were disinfected by wiping with surface disinfectants containing sodium carbonate peroxyhydrate and 1h-benzotriazole as active ingredients (Incidin™Active, 2%ig, 60 min contact time, Ecolab Deutschland GmbH, Monheim am Rhein, Germany) respectively; in the case of sensitive surfaces, a double wipe disinfection with Incidin OxyWipe-S wipes containing hydrogen peroxide and glycolic acid as active ingredients (Ecolab Deutschland GmbH) took place as well as post-cleaning with surfactant-containing water to remove disinfectant residues, all performed by state-certified disinfectors of Charité Facility Management GmbH.

Gassing

For complete pathogen destruction to allow safe cleaning of the rooms, the facility was disinfected via H₂O₂ fumigation by a certified service provider (HIB, Berlin, Germany) based on a decontamination concept specially prepared for this purpose and a risk assessment for the execution of the decontamination work in accordance with the Biological Substances Ordinance and the IfSG (Biosafety Consultant, MLT Medizin- und Labortechnik, Frankfurt am Main, Germany), Technical Rules for Biological Agents No. 120 for ‘Laboratory Animal Science’ and recommendations of the Committee on Hygiene of the Society for Laboratory Animal Science (GV-SOLAS), for disinfection of animal rooms using formaldehyde or hydrogen peroxide (Supplement 8).

Establishment of a crisis team

Immediately after the zoonosis quality of the event became obvious, an operational and an extended crisis team were established. With both teams, it was possible to react to new developments in a timely manner and to deal with various parallel strands of action. This operational crisis team comprised an internal, operationally oriented group of people, consisting of the facility management, the person responsible for breeding and husbandry, the supervisor for animal care and employees of the veterinary service to make organizational preparations for containment, quarantine, diagnostics, hygiene, cleaning and disinfection measures, animal welfare, operational adjustments including the necessary employee briefings and direct communication with authorities. Depending on the facility size, an extended crisis team is to include authorized staff from the areas of facility hygiene, occupational medicine, facility management, occupational safety, biological safety, disaster control, legal department. Corporate communications approved the measures, provided legal and external resources, supervised official coordination with the supervisory authorities, press communications and informed the facility’s management.

Time sequence of the outbreak event

To streamline crisis management all actions taken during the event were assigned to three defined stages of action: ‘detection’, ‘combat’ and ‘review’ (Figure 2).

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Figure 2.

Target/ideal process of an outbreak in the form of a flow diagram.

‘Detection’ comprises activities related to the detection of pathogens in livestock and among contact personnel. In the ‘combat’ stage all disinfection measures for inactivation of infectious pathogens are summarized, including necessary euthanasia of livestock. The ‘review’ stage includes activities to verify prior stage actions and prepare the restart of operations, including follow-up detection.

Without prior knowledge and consistent application of this stage system, further sampling and decontamination measures were carried out at different times in the present case, which must be clearly separated from one another spatially and are associated with additional time and cost (Figure 1).

Environmental samples

Routine sampling of animal rooms for Cb DNA was negative four months prior to the outbreak.

In environmental samples from the holding areas of the affected sheep group, Cb-specific nucleic acid sequences were detected in seven of seven submitted dust samples (Ct values 24.81 to 36.08) and in the submitted litter sample (Ct value 29.73). For polymerase chain reaction (PCR) detection of DNA, a Ct value up to 45 is considered positive. Since cultivation of the pathogen is lengthy and uncertain, the pathogen was detected in coordination with state reference laboratories in further course, mainly via DNA from environmental samples.

In the second environmental sampling (Figure 1), from the corridors, five of 12 dust samples were positive (Ct values 35 to 39). Subsequent sampling of all rooms in the department showed positive results in 25 of 84 samples distributed over the entire department (Ct values 28.96 to 41.12).

Contact persons

Twenty-one per cent (23/110) were positive with positive IgG/IgM antibody titres and acute infection confirmed by further follow-up. This group was directly exposed to the source of infection, that is, they had direct animal contact or were in the immediate vicinity of affected animals.

In 34.5% (38/110), positive IgG antibody titres had been found, with no relevant change in titre during the course, indicating previous infections, mostly unknown to the individuals, interpreted as serum scars. The proportion of the contacts who had no antibodies and remained seronegative was 44.5%.

All persons who tested positive were suspected by the LGA of having an acute infection and were advised to consult an infectious disease specialist to begin treatment as soon as possible. For this purpose, an offer was made to present oneself directly in-house. This was done in collaboration between the Occupational Medicine Centre, the Tropical Institute and the Infectious Diseases Clinic.

Large animals

In the first sampling of the experimental animals (Figure 1), the vaginal swab sample from the ewe tested positive for Cb (Ct value 33.59).

In the third sampling (Figure 1), no pathogen could be obtained by means of fur swab (pigs) and vaginal swab samples (sheep).

Small animals

In more extensive studies of the small animals in the third sampling (Figure 1), Cb DNA (Ct values 34 to 38) was detected in two of seven dust swab samples from the IVC systems and LAF enclosures and in eight of 29 pooled fur swabs (one pooled fur swab from all animals on an animal housing shelf) from the entire small animal population (mice, rats, guinea pigs and rabbits). Neither Q fever antibodies nor Cb-specific nucleic acid sequences were detected in the 52 blood samples and 16 spleen samples from the sentinel animals (FLI). Similarly, no detection was possible in 22 organ pool samples of spleen, lung and liver (LAVES).

Organizational measures after an outbreak of infection

To prevent pathogen spread, affected animal rooms were subjected to relatively negative air pressure compared with surrounding areas controlled by the ventilation system. Samples taken from filter stages in the exhaust air duct of the ventilation and air-conditioning system provided evidence that the pathogen had not spread to the outside environment.

Spread within the building through recirculation in the ventilation system (circulating air operation) could also be excluded, since owing to hygienic standards these systems are 100% fresh air systems.

Even before results were available the veterinary authority ordered that the sheep in question be returned to the farm of origin, as Q fever is a purely surveillance and notifiable animal category E disease (according to the categories of listed diseases under Regulation 2018/1882) without animal-related need for action.

After confirmation of the suspected Q fever (LAVES), the holding rooms of the affected sheep in the large animal husbandry area were quarantined by the responsible health department and the rooms were then cleaned and disinfected as described.

In coordination with animal welfare and occupational safety monitoring authorities, the remaining small animal population was euthanized due to the residual risk of infection to humans following department-wide detection of Cb DNA.

Cleaning, disinfection work

An effective room fumigation was demonstrated for all rooms by bioindicators and chemical indicators. The effective process was confirmed for all foam- or wipe-disinfected rooms by a subsequent, twice-negative examination of dust samples. One complete cleaning round, with sampling and processing samples in the lab averaged out to 3–4 weeks. In the case of positive results, this time-consuming process required variable consecutive rounds of cleaning and sampling of the rooms concerned to achieve two consecutive negative results. Due to the great heterogeneity of surfaces to be cleaned, which in many cases were not smooth, but rough and fissured, inactive residual DNA of the pathogen was to be expected, which further led to positive PCR results. All of this made up to eight consecutive rounds in the worst case of cleaning, sampling and testing necessary.

To further support pathogen DNA degradation for negative PCR detection, all rooms were treated by robotic ultraviolet treatment (UVD ROBOTER MODEL C, UVD Robots® ApS, 5260 Odense S, Denmark).

Revision of the operating protocols of the department

The following innovations were included in the department’s revised operating protocols:

A project-specific risk assessment and an assessment of required protection levels for large animal projects involving farm animals. For example, projects with direct access to the gravid ovine reproductive tract are excluded from further implementation at this site if there is a zoonotic risk within the animal population. Such projects refer to facilities with a higher protection level. This does not apply to minipigs and small animals, since these are sourced from controlled and standardized laboratory animal breeders.

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Figure 3.

Flowchart of critical incidents and accidents according to §13 para. 1 Biostoffverordnung. BSO: Biological Safety Officer; PPE: personal protective equipment

Sourcing requirements for obtaining and maintaining farm animals for use in animal experiments were expanded following the outbreak:

Sourcing of large animals is restricted to the animal husbandry department from approved farms of origin only. Criteria for approval include species-appropriate husbandry, delivery in compliance with animal welfare requirements, possible boarding of laboratory animals with on-site veterinary care, controlled purchase of individual, tested breeding animals and thus traceable supply chains without contact with external animal stocks.

Follow-up

Following two negative pathogen tests and submission of the updated operating protocol, which, in contrast to the previous situation, provides for a strict separation between the large and small animal areas, permission to restart operations was granted by the competent authority.

Six-month follow-up control of all rooms for Cb DNA (LAVES) produced only one positive result out of 109 samples (Ct value 35.89) and was interpreted as inactive residual pathogen signal.