Human and Dromedary Camel Infection with Camelpox Virus in Eastern Sudan

Abstract

We provide evidence for the zoonotic nature of camelpox virus by reporting infections that involved dromedary camels and three camel herders in Showak area of eastern Sudan between September and December 2014. The skin lesions in the camel herders consisted of erythema, vesicles, and pustules that involved arms, hands, legs, back, and abdomen and resolved within less than 2 months with no human-to-human transmission. The diagnosis was achieved through molecular technique, virus isolation in cell culture, and partial genome sequencing.

Introduction

The past two decades have seen an expanded rate of outbreaks of animal and human orthopoxvirus (OPV) infections. These include infections by Vaccinia virus (VACV), Cowpox virus, Monkeypox virus, Buffalopox virus and Camelpox virus (CMLV) (Shchelkunov 2013). Camelpox is a very infectious viral skin disease that occurs in almost every country that practice camel husbandry aside from Australia. The disease is endemic and epidemic in Sudan and a pattern of sporadic epidemics occurs every 2–4 years with an ascent in seasonal incidence usually amid the rainy season (Khalafalla and Mohamed 1996).

It has been almost more than 100 years since camelpox was initially described in Punjab, India, in 1909, yet the zoonotic nature of the CMLV remained an open debate, and a firm articulation that CMLV is pathogenic in man has not been accomplished. The first report of a case of human camelpox was described in Somalia in a 40-year-old camel herder (Kritz 1982); however, the association of CMLV in the affected individual could not be illustrated (Kritz 1982, Jezek et al. 1983). The sparseness of human CMLV infection and probably other OPVs from the 1970s until recently may be because of the global small pox vaccination campaign that ended in the late 1970s. It was in this way hypothesized that human camelpox may turn out to be more common as the immunity of the human population wanes (Baxby 1972, Duraffour et al. 2011). The first indisputable proof of zoonotic CMLV infection in humans connected with outbreaks in dromedary camels has been recently reported in India where three human cases of camelpox have been accounted for (Bera et al. 2011).

In the present communication, we describe cases of camelpox and related cases of human infection in four camel herders at Showak area, Gedarif State of eastern Sudan.

The Study

The Veterinary Research Institute Ethics Review Committee, Sudan, approved this study and an informal consent was obtained from individuals to collect skin swabs.

In August 2014, typical clinical signs of camelpox as previously depicted (Khalafalla and Mohamed 1996) were seen in two migratory herds of dromedary camels near Showak city, Gedarif State, Sudan. Tissue samples of skin scabs were collected from two young dromedaries from each herd (Samples C1 and C2).

In September 2014, a case of skin rash was noted in a 45-year-old male camel herder. The second, third, and fourth human cases were recorded during November–December 2014 in three camel herders aged 19 (case 2), 32 (case 3), and 39 years old (case 4). The four camel herders were not caring for the camels that were sampled in the study, but different camel herds. They did not seek medical treatment and reported no history of vaccination against small pox in childhood. All affected camel herders reported contact with camel herds affected with camelpox and two of them (cases 2 and 4) reported treating and expelling pox lesions from affected camels 2–3 weeks before developing skin rashes. In all the mentioned four human cases, the lesion began as erythema, accompanied by slight fever, itching, and malaise. The nodular lesion took 7–10 days to form into vesicles that later changed to pustules and scabs involving arms, hands, legs, back, and abdomen (Fig. 1), followed by complete healing within 30–40 days except in case 4 who reported an extended recovery period of 4 months. No human-to-human spread of the disease was reported during this study. The skin rash described in this report is similar to that in the reference photo (Bera et al. 2011).

FIG. 1.

Camelpox virus infection in four camel herders who were in contact with affected dromedaries, Showak area, Sudan, 2014. Skin lesions on hand (A) and back (B) of case 2 and hand of case 4 (C) are shown

Sterile cotton swabs were used to collect samples from the pox-like lesions of the four camel herders (Samples H1–H4) by the medical staff of Showak General Hospital. The swabs were preserved in a cell culture media supplemented with antibiotics and transported in the presence of cool packs to the laboratory in Khartoum. Swabs from human cases and tissue scabs from camels were processed for virus isolation in Vero cell culture (Khalafalla et al. 1998) and for PCR. Using a multiplex gel-based PCR (Khalafalla et al. 2015), positive amplification was achieved in both the two camel samples (Sample C1 and C2) and three out of four human samples (Samples H1, H2, and H4). The results were affirmed by a quantitative RT-PCR assay specific for CMLV (Venkatesan et al. 2012). Virus isolation was achieved in one out of three human samples (collected from case 4) and the two camel samples. Cytopathic effects characterized by cell rounding and multinucleated giant cell formation similar to those described previously (Khalafalla et al. 1998) were observed.

A gel-based PCR targeting the A-type inclusion body protein gene (Meyer et al. 1997) was performed using DNA extracted directly from the camel scabs and the human swabs. This PCR was also performed using lysate of cell culture isolates from passage two and the PCR products were sequenced. We performed a phylogenetic analysis of the three sequences together with six homologous sequences obtained from the literature (Table 1). Results of Basic Local Alignment Tool (BLAST) performed using BLASTN 2.5.0 package (Stephen et al. 1997) identified CMLV in all the three examined sequences, and nucleotide sequence alignment showed that the two camel isolates (CMLV-41 and CMLV-43 obtained from samples C1 and C2, respectively) are 99.88% identical with a T deletion mutation in position 6 of the CMLV-41 sequence. In contrast, the human isolate (CMLV-1 SD obtained from sample H4) varied from the two camel isolates in just one position (mutation A454G). The results of the percentage identity matrix (Table 2) revealed that the three CMLV isolates examined in this study share 99.05–99.76% nucleotide sequence identity with the four published CMLV isolates (CMLV-CMS, CMLV-M-96, Orthopox cameli, and CMLV-0408151v) and 94.74–97.07% identity with other members of the genus OPV. In particular, the human isolate CMLV-1 is closer to CMLVs than other OPVs (VACV WR/AY243312, horsepox virus isolate MNR-76/DQ792504, and rabbitpox virus/AY484669) (Table 2).

Table 1.

Information on Camelpox Viruses Isolated from a Camel Herder and Two Dromedary Camels in 2014 in Showak, Sudan, and Homologous Sequences Obtained from the GenBank and Used for the Phylogenetic Analysis of the ATPI Gene

No.	Virus identification	Host	Geographic origin	GenBank acc. no.	Reference
1	CMLV-1	Human	Sudan	KT931623	This study
2	CMLV-41	Dromedary	Sudan	KT931624	This study
3	CMLV-43	Dromedary	Sudan	KT931625	This study
4	CMLV 0408151v	Unknown	Unknown	KP768318	Unpublished
5	CMLV-CMS	Dromedary	Iran	AY009089	Gubser and Smith (2002)
6	CMLV-M-96	Dromedary	Kazakhstan	AF438165	Afonso et al. (2002)
7	Orthopoxvirus cameli	Dromedary	Iran	X69774	Meyer and Rziha (1993)
8	CMLV-Al-Ahsaa 1	Dromedary	Saudi Arabia	JQ901104	Yousif and Al-Naeem (2012)
9	CMLV-Al-Ahsaa 2	Dromedary	Saudi Arabia	GU937751	Yousif et al. (2010)

Table 2.

Percentage Identity of CMLV Isolates and Selected Orthopoxviruses Based on ATI Protein Gene Nucleotide Sequences Obtained by BLASTN 2.5.0

Strain/accession number CMLV isolate	CMLV-41/Showak/2014	CMLV-43/Showak/2014	CMLV-CMS/AY009089	CMLV strain/0408151v KP768318	CMLV M-96/AF438165	Orthopoxvirus cameli/X69774	VACV WR/AY243312	Horsepox virus isolate MNR-76/DQ792504	Rabbitpox virus/AY484669	Taterapox virus strain Dahomy 1968/DP437594	Cowpox virus strain RatKre/KC813505	Buffalopox virus isolate 91222/GU223072
CMLV-1/Human/Showak/2014	99.99	99.88	99.76	99.76	99.76	99.05	96.87	96.87	96.54	96.21	95.40	94.74
CMLV-41/Showak/2014		99.88	99.52	99.52	99.52	99.05	97.09	97.08	96.76	96.28	95.49	95.04
CMLV-43/Showak/2014	99.88		99.53	99.53	99.53	99.05	97.09	97.08	96.76	96.28	95.46	95.04

Phylogenetic analysis (Fig. 2) using the neighbor-joining method in MEGA6 program (Tamura et al. 2013) revealed that the three Sudanese CMLVs clustered together with CMLV-CMS, CMLV-M-96, and CMLV strain 408151v in one branch, whereas the three other virus sequences lay on the alternate branch. All sample preparation, cell culture inoculation, and nucleic acid handling and amplifications were carried out under aseptic techniques to avoid any cross-contamination. Sequences obtained in this study were submitted to GenBank under accession numbers KT931623, KT931624, and KT931625.

FIG. 2.

Phylogeny of camelpox viruses isolated from human and dromedary camels in Sudan in 2014 in comparison with homologous amino acid sequences of the ATP gene obtained from the GenBank. The phylogeny was inferred using the neighbor-joining method. The optimal tree with the sum of branch length = 2.30600510 is shown. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. Evolutionary analyses were conducted in MEGA6. Bootstraps values >70% are indicated (1000 replicates).

No human-to-human spread of camelpox was seen during this limited investigation. In any case, people working as camel herders, traditional healers, vets, and technicians treating or collecting skin samples in endemic areas and laboratory workers who handle CMLV might be at risk for camelpox infection.

Conclusions

We reported an outbreak of camelpox involving two camel herds at Showak area of eastern Sudan and human infection in three male camel herders who had direct contact with affected dromedary camels. Epidemiological data and the genomic sequences suggest possible zoonotic transmission of CMLV from camels to people, but no human-to-human spread was noted. With these verified cases of camelpox in humans, the host range of the causative virus that was considered to solely infect camels should include humans as well. In addition, our findings point that camelpox may become of public health importance and, therefore, more information on its prevalence in humans and dromedary camels is required. Future plans should also include epidemiological surveillance applied by public health authorities in endemic areas to create prevention strategies to avoid virus transmission among individuals who are at risk of infection.

Footnotes

Acknowledgments

We thank Abdo Bushara and the staff of Showak General Hospital for the assistance in sample collection.

Author Disclosure Statement

No competing financial interests exist.

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