Identification of Coxiella burnetii in Tank Raw Cow Milk: First Findings from Chile

Introduction

C oxiella burnetii is a Gram-negative obligate intracellular coccobacillus that causes Q fever, a zoonotic disease of worldwide distribution, except for New Zealand (Keshavamurthy et al. 2019). Many animal species are susceptible to infection but is more prevalent in ruminants (Banazis et al. 2010), causing an economic impact due to abortions, stillbirth, weak calf delivery, metritis, and infertility (Kim et al. 2005). Infection in humans is associated with inhalation of contaminated aerosols generated during parturition, slaughter, feces, urine, and drinking raw milk (Seo et al. 2018, Keshavamurthy et al. 2019), producing a self-limited acute disease, pneumonia, hepatitis, chronic endocarditis in immunocompromised patients, and abortions and stillbirth in pregnant women (Masala et al. 2004). Recent studies have shown an increased prevalence of Q fever in livestock populations around the world (Keshavamurthy et al. 2019).

In South America, no systematic studies have been carried out, with outbreak reports described in French Guyana (Eldin et al. 2014) and Brazil (Costa et al. 2006). In Chile, only one official outbreak of Q fever was declared in August 1998, comprising eight human cases, all staff from the Quarantine Area of the Agriculture and Livestock Service who were in contact with sheep from Spain. Recently, a first epidemiologic study on human Q fever in Chile indicated absence of the disease (Weitzel et al. 2016). In 2017, a human Q fever outbreak was declared in Los Lagos and Los Ríos district in southern Chile. This outbreak produced cases with severe pneumonia in dairy farm workers and families and also health personnel, suggesting possible person-to-person transmission. The objective of this study was to evaluate the presence of C. burnetii in bulk tank milk from dairy farms during the 2017 outbreak in Chile.

Materials and Methods

Raw tank milk samples (N = 105) were obtained from 105 different dairy farms from clinically healthy dairy cows of the Metropolitan, Ñuble, Bío-Bío, Araucanía, Los Ríos, and Los Lagos Districts between February and March 2017 (Fig. 1). DNA was extracted using the Milk Bacterial DNA Isolation kit (Norgen Biotek Corporation, Ontario, Canada) and quantified using Nanodrop 1000 (Thermo Scientific).

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FIG. 1.

Map of Chile district showing the regions where bovine bulk tank milk samples were collected from dairy cattle farm.

Detection of C. burnetii was carried out using a duplex qPCR amplifying two genes targets together. The transposase IS1111 and the outer membrane protein (com1) genes were used as target genes, following protocols suggested by previous studies (Banazis et al. 2010, Lockhart et al. 2011). All reactions were performed in duplicate using the recommendations of the Australian Rickettsial Reference Laboratory. A commercial C. burnetii positive control was included (Ampli Run, Vircell DNA Control^®, Spain). Amplification was performed in an ABI 7500 Fast Real Time PCR System (Applied Biosystems). Sanger DNA sequencing was performed for IS1111 sequences in an AB3500 instrument (Applied Biosystems) using the same primers (Banazis et al. 2010), and identities with published sequences were determined through the nucleotide BLAST tool.

Results and Discussion

Two samples (2.1%) were positive for C. burnetii. One sample (L1) was obtained from the Bío Bío District and one sample (L2) came from Los Lagos District, ∼400 km away. Ct values of 33.6 (sample L1) and 32.4 (sample L2) for com1 gene and 34.4 (sample L1) and 32.1 (sample L2) for IS1111 gene were obtained. The IS1111 DNA sequences were identical to a previously reported gene (GenBank accession no. KT391017.1), confirming the detection of C. burnetti in milk samples.

This is the first study in Chile that identified C. burnetii in bulk tank raw milk samples from dairy farms. The milk came from clinically healthy cows, indicating a possible shedding in milk of C. burnetii asymptomatic animals, thus milk may be a source of infection for humans as is suggested by Kim et al., 2005, and Seo et al., 2018. All positive milk samples were subsequently sold directly to the local community and consumed either raw or boiled, and the farm workers did not use appropriate biosecurity measures, as is common in the majority of small producers in Chile.

Only two time-distant reports of human Q fever have been documented in Chile, one in 1998 and another in 2017, mainly because of the lack of epidemiological surveillance, and the only serological study in humans did not consider the risk groups (Weitzel et al. 2016). The real prevalence of C. burnetii infection in cattle in Chile is unknown, due to lack of surveillance, low access to diagnostic tools, and no epidemiological studies.

The results are consistent with previous studies of Klee et al. (2006) who developed a high sensitive and specific qPCR method to detect C. burnetti in pooled samples, like bulk tank milk in dairy cattle, and has been suggested to be an effective method to investigate the epidemiological condition of the pathogen (Kim et al. 2005, Lockhart et al. 2011). Bulk tank milk has been used for surveillance of other bovine diseases and could be used also for Q fever.

Conclusions

This is the first report using qPCR to detect C. burnetti in bulk tank milk samples in dairy cows in Chile. Our results indicated a wide distribution (400 km) with a very low incidence (2.1% of the samples) but potentially transmissible to humans. A larger epidemiological study is needed to have a better understanding of the epidemiological situation of the disease, including animal and environmental sources of Q fever locally, because it is essential to improve prevention and control of this disease in Chile and South America.