Leptospirosis in animals and human contacts in Egypt : broad range surveillance

Introduction: Leptospirosis is a re-emerging zoonotic disease of humans and animals worldwide. The disease is caused by pathogenic species of the genus Leptospira. These organisms are maintained in nature via chronic renal infection of carrier animals, which excrete the organisms in their urine. Humans become infected through direct or indirect exposure to infected animals and their urine or through contact with contaminated water and soil. This study was conducted to investigate Leptospira infections as a re-emerging zoonosis that has been neglected in Egypt. Methods: Samples from 1,250 animals (270 rats, 168 dogs, 625 cows, 26 buffaloes, 99 sheep, 14 horses, 26 donkeys and 22 camels), 175 human contacts and 45 water sources were collected from different governorates in Egypt. The samples were collected from different body sites and prepared for culture, PCR and the microscopic agglutination test (MAT). Results: The isolation rates of Leptospira serovars were 6.9%, 11.3% and 1.1% for rats, dogs and cows, respectively, whereas the PCR results revealed respective detection rates of 24%, 11.3% and 1.1% for rats, dogs and cows. Neither the other examined animal species nor humans yielded positive results via these two techniques. Only six Leptospira serovars (Icterohaemorrhagiae, Pomona, Canicola, Grippotyphosa, Celledoni and Pyrogenes) could be isolated from rats, dogs and cows. Moreover, the seroprevalence of leptospiral antibodies among the examined humans determined using MAT was 49.7%. Conclusions: The obtained results revealed that rats, dogs and cows were the most important animal reservoirs for leptospirosis in Egypt, and the high seroprevalence among human contacts highlights the public health implications of this neglected zoonosis.

Leptospirosis is a reemerging zoonotic disease of humans and animals worldwide.It is presumed to be the most widespread zoonosis in the world.The disease is caused by pathogenic species of spirochetes of the genus Leptospira.The organisms are maintained in nature by chronic renal infection of carrier animals, which excrete the organisms in their urine.Humans become infected through direct exposure to infected animals or their urine or through indirect contact with contaminated water or soil (1) .Animal species that serve as a maintenance host for one serovar can become an accidental host of another serovar.Moreover, every mammalian species is potentially an accidental host for leptospirosis, and no species has been identifi ed as refractory to infection.Currently, there are more than 250 known serovars of Leptospira spp., which show a variable distribution by geographical region and host species (2) .
In developing countries, agriculture continues to be the main source of employment and income for a large number of rural residents.Living and working in close contact with animals or their waste may present greater opportunities for exposure and infection (3) .
Few data are available on the incidence and prevalence of leptospirosis in the Middle East (1) .In Egypt, a pilot study conducted approximately 25 years ago revealed that 9% of sera collected from persons living in contact with carrier animals were seropositive for different Leptospira serogroups (4) , and an earlier study (5) reported that 13% of the collected rodent sera were reactive for leptospirosis.More recent studies have been initiated to detect leptospiral antibodies among undiagnosed acute febrile illness (AFI) and hepatitis patients in Egypt (6) (7) .Approximately 16% of sera from both disease groups showed seroreactivity to Leptospira immunoglobulin M (IgM) by enzymelinked immunosorbent assay (ELISA) and the microscopic agglutination test (MAT).These studies have revealed that the epidemiologic status of leptospirosis in Egypt needs to be defi ned and that diffi culties in disease diagnosis need to be eliminated.Most patients with AFI are currently empirically diagnosed as typhoid or brucellosis cases and treated accordingly with chloramphenicol or tetracycline derivatives (6) .More recently, cross-species surveillance of Leptospira in animals has been carried out in the vicinity of Mahalla, a portion of a governorate of Egypt, to determine the most common serovars affecting this region (3) .The fi ndings of this study suggested that wild and domestic mammals are important sources of pathogenic leptospires in Mahalla City.Additionally, Hatem and Samir (8) , recorded a recent outbreak of leptospirosis in sheep as the fi rst epidemic in northern Egypt.Therefore, the current study involved broad range surveillance of the zoonotic bacterial agent Leptospira in animals and human contacts in Egypt.The study aids in determining the most likely sources of leptospires infecting humans and the distribution of the various serovars in the region.This information is important to determine and optimize appropriate intervention strategies and methods for the diagnosis/management of leptospirosis cases in animals, especially in pet canines and livestock.

METHODS
The study was performed at the Department of Microbiology, Faculty of Veterinary Medicine, Cairo University, Egypt, under the recommendations of the Institutional Animal Care and Use Committee Guidebook.Table 1 shows the different animal species and the different tissues examined in this study.

Study area
The samples were collected from 14 different governorates: Alexandria (representing the coastal area); Gharbeya, Kafr El-Sheikh, Sharkya, Dakahleyya, Menufya, Behera and Qualyobya (representing the Delta); Giza, Fayoum, Beni-Swaif, 'Menia, and Assuit (representing upper Egypt); and El-Wadi El-Gedeed (representing the border area).The animals were randomly selected.Rodents and stray dogs were captured using traps.In the case of livestock, all specimens were collected either from farms, during presentation of the animals at veterinary clinics, or from animals owned by individuals.The examined camels were found in the Southern border areas.

Specimen collection
Blood samples from rats and dogs were obtained via heart puncture, after which the animals were euthanized and subjected to necropsy to collect urine and kidney samples.For the other animals, urine samples were collected via catheterization and blood was then drawn from the jugular vein.

Serum samples
For the animals and human contacts involved in the study, 3 to 5ml of blood was collected in anticoagulant-free tubes and spun down at 5,000rpm for 20 min.All sera were transferred in cryovials and stored at -20°C until use for further serological testing (i.e., MAT).

Water sources
Water samples were collected from small water streams in regions where the examined animals were living in different governorates.

Laboratory procedures
Animals were considered positive for leptospirosis infection if the organism was recovered through culture, and/or polymerase chain reaction (PCR)-specifi c assays for pathogenic Leptospira sp. were positive, whereas a titer ≥ 200 against a pathogenic serovar was considered positive by the MAT.
The collected organs (kidneys) were macerated with sterile blades (or by passage through a 3ml syringe without a needle) and inoculated in the same manner.All cultures were incubated at 30°C for 6-8 weeks and were examined weekly using a dark-fi eld microscope.Positive cultures were purifi ed and subcultured in EMJH.Fletcher's semisolid medium was used for maintenance.

PCR diagnostics
Deoxyribonucleic acid (DNA) was extracted from the inoculated Leptospira culture media using the QIAmp DNA Mini Kit (Qiagen, Valencia, CA) in accordance with the manufacturer's instructions.PCR was carried out via amplifi cation with specifi c primers for pathogenic leptospires (lig1/lig2) according to the protocol previously described by Palaniappan et al (11) .The PCR products were run on agarose gels containing ethidium bromide and visualized with a transilluminator at 468bp (Figure 1).

Microscopic agglutination test
A standard MAT was performed on sera to determine the most reactive Leptospira serogroups.The test was carried out according to the methods described by WHO (12) .Briefl y, live   Leptospira cell suspensions representing 24 serovars (kindly obtained from US Naval Medical Research Unit -3) were added to serially diluted serum specimens in 96-well microtiter plates and incubated at ambient temperature for 1.5 hours.

DISCUSSION
Agglutination was then examined via dark-fi eld microscopy at a magnifi cation of 100X.A reactive MAT was determined by a titer ≥ 200.The reported titers were calculated as the reciprocal of the highest serum dilutions that agglutinated at least 50% of the cells for each tested serovar.Additionally, MAT is a tool through which the recovered isolates can be speciated using 20 different standard antisera.
Various prevalence rates of leptospirosis were determined using the applied culture techniques, PCR and MAT after examination of the collected samples and are shown in Table 2.In addition, the results of speciation analysis revealed that only six serovars (Icterohaemorrhagiae, Pomona, Canicola, Grippotyphosa, Celledoni and Pyrogenes) were the most predominant (Table 3).Moreover, of the 175 examined humans, 87 (49.7%) were serologically positive for Leptospira serovars antibodies, and all of the positive human sera showed reactivity with the isolated animal serovars (Table 4).However, the serological examination of animal sera using the MAT for the detection of specifi c antibodies against different serovars revealed that Leptospira Icterohaemorrhagiae and Leptospira Pomona were the predominant serovars in rats, cows, buffaloes and sheep, whereas Leptospira Icterohaemorrhagiae and Leptospira Canicola were more common among dogs.The serovars isolated from the two positive donkeys were identifi ed as Leptospira Grippotyphosa and Leptospira Celledoni.
Leptospirosis is increasingly being recognized as an important cause of hemorrhagic fever in humans (13) .The classic presentation of severe leptospirosis (Weil's disease) is characterized by jaundice and acute renal failure.Leptospirosis is a worldwide, acute febrile zoonosis.Many animals are susceptible to the disease.The leptospires infecting humans are primarily maintained by warm-blooded vertebrates.In cities, Norway rats (Rattus norvegicus) and dogs have been regarded as major reservoirs.Humans can become infected when they come in contact with such animals or their contaminated excreta (14) .In Egypt, recent surveillance of AFI (acute febrile illness) patients revealed that among the areas tested, Mahalla city showed a higher frequency of leptospirosis compared with other regions of the country (7) (15) .Therefore, approximately four years later, another study was published providing data on the distribution of leptospirosis in animals in Mahalla (3) .Prior to this study, the most recent animal surveys in Egypt were conducted in Cairo over 20 years ago (4) .In the current investigation, isolation was successful only from rats, dogs and cattle (Table 2).The isolates recovered from rats were identifi ed as Icterohaemorrhagiae (n=5), Pomona (n=4), Canicola (n=4), Grippotyphosa (n=3) and Celledoni (n=1), whereas those from dogs were Canicola (n=10) and Icterohaemorrhagiae (n=9), and the cow isolates were Pomona (n=5) and Icterohaemorrhagiae (n=2).
Notably, 9 rat isolates (Icterohaemorrhagiae and Pomona) were recovered from rats captured inside dairy farms.These serovars were also recovered from water sources supplying such farms (5 Pomona isolates and 2 Icterohaemorrhagiae isolates).In addition, the serological survey (MAT) demonstrated that the Icterohaemorrhagiae and Pomona serovars were the most prevalent among rats, followed by Celledoni, Canicola and Grippotyphosa.Icterohaemorrhagiae and Pomona were also the most prevalent serovars among cattle, which is in accord with the results obtained by Hatem et al. (16) , while Icterohaemorrhagiae and Canicola were the most prevalent among dogs.However, the seroprevalence of Leptospira serovars observed in humans who had contact with captured animals showed that Pomona, Canicola and Icterohaemorrhagiae were the most common reactive serovars, followed by Pyrogenes and Grippotyphosa.
Regarding rats, a recent study (3) found that the detection rate of Leptospira antibodies was lower (23%) compared with an earlier study from Egypt (55.4%) (4) and many other reports from different parts of the world (17) (18) (19) .However, the same study (3) reported that dogs had never been evaluated as carriers of Leptospira in Egypt, and the observed seroprevalence in that study was 12%, which was similar to the seroprevalence reported in Italy (20) and Thailand (21) but much lower than that reported in Germany (22) , Mexico (23) and the USA (24) .The same study found a high seroprevalence of leptospires in cows (44%).The authors added that this information supported the conclusions drawn by other researchers, who determined that livestock breeding confers a major occupational risk (1) (25) (26) .Comparatively, in the present study, the seroprevalence of leptospirosis detected in rats, dogs and cows was 75.9%, 58.3% and 37.6%, respectively.
Our results showed that no isolates could be recovered from sheep, although the animals were serologically reactive (approximately 45%) for Icterohaemorrhagiae and Pomona, showing relatively high titers (800 and 6,400, respectively).This fi nding contrasts results previously obtained in Egypt (3) , where sheep samples were found to be negative for leptospirosis by all of the applied diagnostic tools.However, earlier studies conducted in Egypt revealed that 4.2% of sheep were seropositive (25) .This discrepancy may be due to differences in the screened localities, the number of animals used, or the applied laboratory testing methods.Worldwide, the reported prevalence of leptospirosis in sheep has been found to be comparatively low in Canada (27) , Portugal (26) , Chile (28) , Italy (29) , and Brazil (30) .Only 7.7% of the donkey sera collected in this study were reactive for leptospirosis, particularly against Grippotyphosa and Celledoni.However, two earlier studies from Egypt showed that 17-29% of the tested donkeys were seropositive for Leptospira (4) (25) .Another recent study conducted in the Middle East determined that Iranian donkeys and horses showed a high rate of seroconversion (40% and 28%, respectively), particularly against L. interrogans serovars Icterohaemorrhagiae, Ballum and Pomona (31) .In contrast, the horse and camel specimens collected in the current study were negative for the presence of Leptospira cells, DNA and even antibodies indicating infection.
A few previous studies have screened buffaloes for leptospirosis, including an earlier report from Egypt that demonstrated a seroprevalence of 26% (25) .This result is similar to that obtained in a previous study in Mahalla (3) , but it is considerably higher than that reported from South Africa (1.7%) (32) .The present study found that approximately 15% of the collected buffalo sera were reactive to Leptospira antibodies.
The current investigation provides an indication of the current situation regarding the epidemiology of this zoonosis in Egypt.It is clear that leptospirosis has become a problem in large cities in Egypt and is not limited to small areas.Similar studies have confi rmed this prevalence based on surveillance conducted in other regions of the world, such as Brazil (13) .Our results are helpful for determining which isolates can be found in particular animals in Egypt.Furthermore, our serosurvey of humans who were in contact with such animals revealed a high prevalence rate of 49.7%, and Leptospira Pomona, Canicola and Icterohaemorrhagiae were the most prevalent serovars identifi ed in human infections.Interestingly, we were also able to recover Leptospira serovars from water streams; such contaminated water constitutes a potential hazard for humans.
This information is helpful for determining strategies for leptospirosis prevention and control in humans and animals by eradicating the disease in animals (source of infection) via the application of appropriate vaccination programs.A targeted vaccination program for dairy cows and dogs using a multi-serovar vaccine manufactured from the recovered isolates (bivalent or trivalent) would limit the disease burden among livestock and pets, thereby decreasing environmental contamination and accordingly reducing human exposure to this pathogen.
A significant effort is needed to increase community awareness that clean water sources should be employed for drinking, cooking and cleaning, rather than using untreated water directly from canals or streams.Farm laborers should consider the feasibility and practicality of wearing personal protective equipment when performing high-risk tasks.Effective diagnostic methods should be established as needed, as the disease has generally been left undiagnosed and underestimated.
The authors declare that there is no confl ict of interest.

TABLE 2 -Total number of Leptospira-positive cultures compared with PCR-and MAT-positive samples from different sources.
PCR: polymerase chain reaction; MAT: microscopic agglutination test; NA: not applicable.

TABLE 4 -Number of cases positive by MAT correlated with the titers and serovars of the collected human contact sera.
MAT: microscopic agglutination test.

TABLE 3 -Results of serotyping of the isolates recovered from different animal species.
n: number; L: Leptospira.