We chose Cholistan as the study site because it is the most important region of Pakistan for the camel industry, and handlers and their families are in close contact with dromedaries. We engaged study participants in the Bahawalnagar and Bahawalpur districts, located in southern Punjab Province, Pakistan. The Institutional Ethical Review Board (IERB) of the Institute of Public Health, Government of Punjab, Lahore, Pakistan, approved the study. We obtained written informed consent from all study participants.

Camel handlers in Cholistan differ from those in Saudi Arabia in that they own their camels, along with cows, goats, and sheep, and they and their families take care of these animals. Both men and women are responsible for grazing, feeding, milking, and waste disposal. In addition, they live in close proximity to camels and share similar water sources (8–10). Camel handlers in Cholistan are either nomadic, seminomadic, or sedentary, with varying degrees of exposure to camels. Nomads live with their camels in the desert and migrate throughout Cholistan, whereas seminomads tend to live at a base camp and migrate depending on availability of fodder and water. Nomadic camel handlers and their families have the highest exposure to camels, whereas sedentary ones have the least exposure.

During 2017–2018, we obtained blood samples from 100 participants from nomadic, seminomadic, and sedentary populations. The age range was 8–76 years (average 30.1 years). We obtained demographic and clinical information at sampling by written questionnaire, including participant age, lifestyle (nomadic, seminomadic, sedentary), role in family (husband, wife, child, etc.), underlying medical conditions, numbers of camels owned, history of tobacco use (smoking or chewing), and consumption of camel products (milk) (Table 1; Appendix Table). We transported samples to the microbiology department at the University of Veterinary and Animal Sciences (Lahore, Punjab, Pakistan). We prepared serum samples, stored them at −80°C, and shipped them to the University of Iowa (Iowa City, Iowa, USA) for analysis.

We tested all the samples for MERS-CoV–specific antibodies by ELISA and 50% reduction plaque-reduction neutralization test (PRNT₅₀). Of 91 participants examined by a commercially available ELISA, 49 were positive for MERS-CoV–specific antibody. Twelve had PRNT₅₀ titers >1:20 and were considered positive; of these, 5 were also positive by ELISA. In addition, 10/12 were positive by immunofluorescence assay. Of the 12 PRNT₅₀-positive participants, 3 were women and 1 was an 8-year-old child (Table 2).

All but 2 of the study participants were exposed to camels. There was no significant correlation (p>0.5) between MERS-CoV seropositivity and lifestyle, presence of concurrent conditions, drinking unpasteurized camel milk, or tobacco use, with the caveat that the sample size was small.

In general, nomads had the most and sedentary populations had the least camel contact, although nearly all family members were exposed to and took care of camels. Of 100 participants, we identified 12 who were MERS-CoV seropositive, as measured by the presence of PRNT₅₀ antibody. Of note, several PRNT₅₀-positive samples were negative by ELISA, but most were positive by immunofluorescence assay. This lack of concordance between ELISA and PRNT₅₀ titers was observed previously (3,11) and may reflect lower sensitivity of the commercial ELISA kit (12). Other coronaviruses circulate in camel populations (13), and it is conceivable that the high rate of ELISA seropositivity resulted from immune responses to other, possibly MERS-like, coronaviruses present in Pakistan. Thus, it will be important to assess camel (and human) populations for other coronaviruses that might elicit a cross-reactive response.

The mechanism of MERS-CoV transmission from camels to humans in Pakistan is not established, but most camel handlers and their families drink fresh camel milk, obtained after young camels have finished nursing. Juvenile camels demonstrate the highest rate of seroconversion and of MERS-CoV positivity (6,14), so it is possible that drinking fresh milk is a source of infection. In this region of Pakistan, camel handlers and their families also share water sources with camels, which probably contributes to virus transmission. Zohaib et al. identified a 75.6% MERS seroprevalence in camels throughout Pakistan, but 0% seropositivity in humans, including some with camel contact (7).

Medical services in Cholistan and adjacent areas are limited, making MERS diagnosis and transmission studies difficult. Our findings show a need for additional studies to confirm the absence of clinically apparent MERS in this region and to determine whether epidemiologic, technical, or other factors caused differences in seropositivity between our study and that of Zohaib et al.

Our study, by demonstrating a low but detectable rate of MERS-CoV seropositivity in camel handlers and their families, indicates that this population could contribute to MERS-CoV transmission to the broader community in Pakistan. We previously showed that measurement of T cell responses identified additional MERS-CoV–immune persons (3,11), suggesting that our results may underestimate the prevalence of MERS-CoV infection. Our results also illustrate the importance of educating camel herders and their families about proper infection control measures, including handwashing, to diminish the likelihood of MERS-CoV transmission.