Transboundary and Emerging Diseases
ORIGINAL ARTICLE
West Nile Virus Infection in Horses in Jordan: Clinical Cases, Seroprevalence and Risk Factors S. M. Abutarbush and A. M. Al-Majali Department of Veterinary Clinical Sciences, Faculty of Veterinary Medicine, Jordan University of Science and Technology, Irbid, Jordan
Keywords: West Nile virus; horses; Jordan; nervous signs; equine encephalitis Correspondence: S. M. Abutarbush. Department of Veterinary Clinical Sciences, Faculty of Veterinary Medicine, Jordan University of Science and Technology, Irbid 22110, Jordan. Tel.: +962 796 620635; Fax: +962 272 01081; E-mail: [email protected] Received for publication August 18, 2013 doi:10.1111/tbed.12191
Summary The objectives of this study are to report clinical WNV infection in horses and to determine the seroprevalence of and risk factors for WNV infection in horses in Jordan. In late summer and early fall of 2012, two mares were presented for evaluation of neurological signs. The first mare had hind-limb ataxia. The second mare was slightly depressed and lethargic. She had ataxia in her four limbs and cranial nerves deficits. Both horses were found positive for WNV IgM antibodies using commercial IgM-capture ELISA test. Both horses were treated symptomatically and recovered uneventfully. The occurrence of clinical cases initiated the need for a seroprevalence and risk factors study. Two hundred and fifty-three normal horses were randomly enrolled in the study. Enrolled horses were grouped into five major regions according to the geographical proximity and climatic similarities. From each region, around 50 horses were sampled. The serum collected from each horse was screened by a competitive ELISA, and those that reacted positive using the previous ELISA test were further tested using commercial IgM-capture ELISA test. Sixty-three horses (24.9%) of the 253 surveyed were seropositive to WNV. Of the 63 horses, none had IgM antibodies for WNV. The region with the highest prevalence was Jordan Valley and Balqa. Horses used for polo (OR = 9.77; 95%CI = 1.32–25.44) and horses located in Jordan Valley and Balqa region (OR = 13.31; 95% CI = 2.33–32.54) were identified as risk factors for seropositivity to WNV in Jordan. These risk factors were attributed to the hot and humid weather, which enhance vector availability. West Nile virus appears to be endemic in Jordan. Future studies are warranted to evaluate the virus situation in the country during the next few years in an attempt to control it.
Introduction West Nile virus is an arbovirus, arthropod-borne, disease that can cause severe illness in animals and humans (Heinz et al., 2000). Before 1994, WNV was not thought to cause public health problems (Schmidt and Elmansoury, 1963). Since the mid 1990s, the disease has been reported more frequently with serious illness in humans and animals (horses) around the world (Le Guenno et al., 1996; Tber-Aldelhaq, 1996; Tsai et al., 1998; Nur et al., 1999; Cantile et al., 2001; Platonov, 2001; Trock et al., 2001; Weese et al., 2003). When bitten by a mosquito that carry the virus, humans and horses can
become infected, and although in the natural cycle, they do not contribute to the spread or amplification of the virus (Bunning et al., 2002). The majority of WNV infections in horses are asymptomatic; 10% of WNV-infected horses develop clinical disease (Bunning et al., 2002; Long et al., 2003; CastilloOlivares and Wood, 2004). The disease can cause signs that range from mild fever to severe neurological dysfunction (Solomon and Vaughn, 2002). Clinical signs include ataxia, paresis or paralysis of the hind and/or front limbs, recumbency and death. Most neurological deficits are related to lesions in the central nervous system. However, the disease can cause cranial nerves dysfunction and signs
© 2014 Blackwell Verlag GmbH • Transboundary and Emerging Diseases. 61 (Suppl. 1) (2014) 1–6
1
West Nile Virus Infection in Horses in Jordan
S. M. Abutarbush and A. M. Al-Majali
such as facial nerve paralysis, paresis of the tongue and dysphagia. Some affected horses recover completely while others remain with neurological deficits for the rest of their life or die. Sometimes affected horses are euthanized on humane grounds. The disease is diagnosed based on history, season of presentation, clinical signs and laboratory tests. ELISA and PCR are used to confirm the disease. There is no specific treatment for the disease in horses. Supportive treatment is usually implemented to reduce CNS inflammation and oedema, prevent self-inflicted injuries and provide nutritional and fluid requirements. In endemic areas, West Nile virus infection is best prevented by vaccination and vector control programmes. Various vaccines types have been used to protect horses against infection. These include inactivated, DNA, live attenuated and genetically modified live vaccines. The number of affected horses was greatly reduced after the introduction of the inactivated vaccine for horses globally. West Nile virus infection is known to be endemic in the Middle East area (Weinberger et al., 2001). Some studies suggested that WNV introduction and latest outbreaks during the 2000s in North America were caused by a WNV that was imported from the Middle East area (Lanciotti et al., 2002; Charrel et al., 2003). In Jordan, the disease has been studied in humans, and WNV infection in horses has been documented in the neighbouring countries (Batieha et al., 2000 and Weinberger et al., 2001). However, the occurrence of the disease in animals in general and in horses in particular has never been reported in Jordan. The objectives of this study are to report clinical WNV infection in horses and to determine the seroprevalence of and risk factors for WNV infection in horses in Jordan. Clinical Cases In late summer and early fall of 2012, 2- and 5-year-old mares were presented for evaluation of neurological signs that started few days before presentation. The two horses belong to different farms in Madaba province. The first mare was bright, alert and responsive, and no cranial nerve deficits were observed. She had difficulty in standing after lying down and was maintaining dog sitting position for a while before she stands. The front limbs were normal, but she appeared ataxic in her hind feet and had normal defecation and urination. The second mare was slightly depressed, but alert and responsive. She was presented for lethargy and ataxia in her four limbs. Examination of the cranial nerves revealed facial cranial nerve dysfunction. The lower lip was droopy, and saliva was drooling from the mouth. She was severely ataxic 2
and had loss of proprioception in all limbs. Urination and defecation appeared normal. Neurological dysfunctions in the two mares were attributed to central nervous system lesions. Complete blood cell count (CBC) and serum biochemical analysis revealed no significant findings. Both horses were found positive for WNV IgM antibodies using commercial IgM-capture ELISA test (ID Screenâ West Nile IgM Capture; ID Vet, Grabels, France). Both horses were treated with steroidal anti-inflammatory drugs and supportive therapy. They recovered uneventfully within few days. The occurrence of clinical cases, with acute exposure, initiated the need for a seroprevalence study to evaluate the spread of the disease in the horse population, which is the first of its kind in Jordan.
Seroprevalence and Risk Factors Materials and methods Study animal and design Horse population of Jordan, as reported by the latest statistical data from Jordan Ministry of Agriculture, is estimated to be 2182 horses (Department of Statistics, 2007). Two hundred and fifty-three horses were randomly enrolled in the study. Simple randomization technique was applied with stratification to geographical location. Stables and horse holdings were selected based on data obtained from the Ministry of Agriculture. Owners were asked to select horses to be enrolled in the study before revealing the study objectives. The horses were clinically normal and distributed over all provinces of Jordan. Enrolled horses from the different provinces were grouped into five major regions according to the geographical proximity and climatic similarities. Those were (i) Irbid, Ajlun and Jerash, (ii) Amman and Madaba, (iii) Ma’an, Karak, Tafelah and Aqaba, (iv) Mafraq and Zarka and (v) Jordan Valley and Balqa. From each region, around 50 horses were sampled. Data collection For each horse, a form was completed and included information about location, age, sex, breed, use of horse, type of nutrition (grazing or not), time owned by the owner and herd size. The age was classified into three groups; 10 years. The time, during which the owner has had the horse for, has been classified into two groups; 1 year. The herd size was further grouped into three groups; 40 horses. Blood samples A venous blood sample was collected from the jugular vein of each horse. The blood was collected into a plain tube,
© 2014 Blackwell Verlag GmbH • Transboundary and Emerging Diseases. 61 (Suppl. 1) (2014) 1–6
S. M. Abutarbush and A. M. Al-Majali
West Nile Virus Infection in Horses in Jordan
which was centrifuged. After harvesting the serum, it was stored at 20°C until it is used for serology. Serology The serum samples of all horses were screened for WNV antibodies using a commercial competitive ELISA test (ID Screenâ West Nile Competition; ID Vet), according to the manufacturer’s instructions. This competitive ELISA detects antibodies directed against the envelop protein of the West Nile virus. Serum samples that reacted positive using the previous ELISA test were further tested using commercial IgMcapture ELISA test (ID Screenâ West Nile IgM Capture; ID Vet), according to the manufacturer’s instructions. This ELISA detects anti-envelope protein IgM antibodies for West Nile virus in horses. According to the manufacturer, the sensitivity and specificity for both ELISAs are 100%. Statistical analysis Initially, univariable analysis was carried out by chi-square analysis. Variable were further assessed using a multivariable logistic regression model. All variables with P < 0.20 (two-sided) in the univariable analysis were further tested by the model. Variables were excluded from the model using a backward-stepwise approach. Statistical analyses were performed using SPSS software, version 17 (SPSS Inc., Chicago, IL, USA). Results Using the competitive ELISA test, 63 horses (n = 63, 24.9%) of the 253 surveyed were seropositive to WNV. Of the 63 horses, none had IgM antibodies for WNV. The distribution of the positive horses in Jordan is shown in Fig. 1. The region with the highest prevalence was Jordan Valley and Balqa, followed by Mafraq and Zarka, Ma’an, Karak, Tafelah and Aqaba, Amman and Madaba, and Irbid, Ajlun and Jerash, respectively. The chi-square univariable analysis revealed five factors (herd size, location, sex, use of the horse and grazing) with P value
ORIGINAL ARTICLE
West Nile Virus Infection in Horses in Jordan: Clinical Cases, Seroprevalence and Risk Factors S. M. Abutarbush and A. M. Al-Majali Department of Veterinary Clinical Sciences, Faculty of Veterinary Medicine, Jordan University of Science and Technology, Irbid, Jordan
Keywords: West Nile virus; horses; Jordan; nervous signs; equine encephalitis Correspondence: S. M. Abutarbush. Department of Veterinary Clinical Sciences, Faculty of Veterinary Medicine, Jordan University of Science and Technology, Irbid 22110, Jordan. Tel.: +962 796 620635; Fax: +962 272 01081; E-mail: [email protected] Received for publication August 18, 2013 doi:10.1111/tbed.12191
Summary The objectives of this study are to report clinical WNV infection in horses and to determine the seroprevalence of and risk factors for WNV infection in horses in Jordan. In late summer and early fall of 2012, two mares were presented for evaluation of neurological signs. The first mare had hind-limb ataxia. The second mare was slightly depressed and lethargic. She had ataxia in her four limbs and cranial nerves deficits. Both horses were found positive for WNV IgM antibodies using commercial IgM-capture ELISA test. Both horses were treated symptomatically and recovered uneventfully. The occurrence of clinical cases initiated the need for a seroprevalence and risk factors study. Two hundred and fifty-three normal horses were randomly enrolled in the study. Enrolled horses were grouped into five major regions according to the geographical proximity and climatic similarities. From each region, around 50 horses were sampled. The serum collected from each horse was screened by a competitive ELISA, and those that reacted positive using the previous ELISA test were further tested using commercial IgM-capture ELISA test. Sixty-three horses (24.9%) of the 253 surveyed were seropositive to WNV. Of the 63 horses, none had IgM antibodies for WNV. The region with the highest prevalence was Jordan Valley and Balqa. Horses used for polo (OR = 9.77; 95%CI = 1.32–25.44) and horses located in Jordan Valley and Balqa region (OR = 13.31; 95% CI = 2.33–32.54) were identified as risk factors for seropositivity to WNV in Jordan. These risk factors were attributed to the hot and humid weather, which enhance vector availability. West Nile virus appears to be endemic in Jordan. Future studies are warranted to evaluate the virus situation in the country during the next few years in an attempt to control it.
Introduction West Nile virus is an arbovirus, arthropod-borne, disease that can cause severe illness in animals and humans (Heinz et al., 2000). Before 1994, WNV was not thought to cause public health problems (Schmidt and Elmansoury, 1963). Since the mid 1990s, the disease has been reported more frequently with serious illness in humans and animals (horses) around the world (Le Guenno et al., 1996; Tber-Aldelhaq, 1996; Tsai et al., 1998; Nur et al., 1999; Cantile et al., 2001; Platonov, 2001; Trock et al., 2001; Weese et al., 2003). When bitten by a mosquito that carry the virus, humans and horses can
become infected, and although in the natural cycle, they do not contribute to the spread or amplification of the virus (Bunning et al., 2002). The majority of WNV infections in horses are asymptomatic; 10% of WNV-infected horses develop clinical disease (Bunning et al., 2002; Long et al., 2003; CastilloOlivares and Wood, 2004). The disease can cause signs that range from mild fever to severe neurological dysfunction (Solomon and Vaughn, 2002). Clinical signs include ataxia, paresis or paralysis of the hind and/or front limbs, recumbency and death. Most neurological deficits are related to lesions in the central nervous system. However, the disease can cause cranial nerves dysfunction and signs
© 2014 Blackwell Verlag GmbH • Transboundary and Emerging Diseases. 61 (Suppl. 1) (2014) 1–6
1
West Nile Virus Infection in Horses in Jordan
S. M. Abutarbush and A. M. Al-Majali
such as facial nerve paralysis, paresis of the tongue and dysphagia. Some affected horses recover completely while others remain with neurological deficits for the rest of their life or die. Sometimes affected horses are euthanized on humane grounds. The disease is diagnosed based on history, season of presentation, clinical signs and laboratory tests. ELISA and PCR are used to confirm the disease. There is no specific treatment for the disease in horses. Supportive treatment is usually implemented to reduce CNS inflammation and oedema, prevent self-inflicted injuries and provide nutritional and fluid requirements. In endemic areas, West Nile virus infection is best prevented by vaccination and vector control programmes. Various vaccines types have been used to protect horses against infection. These include inactivated, DNA, live attenuated and genetically modified live vaccines. The number of affected horses was greatly reduced after the introduction of the inactivated vaccine for horses globally. West Nile virus infection is known to be endemic in the Middle East area (Weinberger et al., 2001). Some studies suggested that WNV introduction and latest outbreaks during the 2000s in North America were caused by a WNV that was imported from the Middle East area (Lanciotti et al., 2002; Charrel et al., 2003). In Jordan, the disease has been studied in humans, and WNV infection in horses has been documented in the neighbouring countries (Batieha et al., 2000 and Weinberger et al., 2001). However, the occurrence of the disease in animals in general and in horses in particular has never been reported in Jordan. The objectives of this study are to report clinical WNV infection in horses and to determine the seroprevalence of and risk factors for WNV infection in horses in Jordan. Clinical Cases In late summer and early fall of 2012, 2- and 5-year-old mares were presented for evaluation of neurological signs that started few days before presentation. The two horses belong to different farms in Madaba province. The first mare was bright, alert and responsive, and no cranial nerve deficits were observed. She had difficulty in standing after lying down and was maintaining dog sitting position for a while before she stands. The front limbs were normal, but she appeared ataxic in her hind feet and had normal defecation and urination. The second mare was slightly depressed, but alert and responsive. She was presented for lethargy and ataxia in her four limbs. Examination of the cranial nerves revealed facial cranial nerve dysfunction. The lower lip was droopy, and saliva was drooling from the mouth. She was severely ataxic 2
and had loss of proprioception in all limbs. Urination and defecation appeared normal. Neurological dysfunctions in the two mares were attributed to central nervous system lesions. Complete blood cell count (CBC) and serum biochemical analysis revealed no significant findings. Both horses were found positive for WNV IgM antibodies using commercial IgM-capture ELISA test (ID Screenâ West Nile IgM Capture; ID Vet, Grabels, France). Both horses were treated with steroidal anti-inflammatory drugs and supportive therapy. They recovered uneventfully within few days. The occurrence of clinical cases, with acute exposure, initiated the need for a seroprevalence study to evaluate the spread of the disease in the horse population, which is the first of its kind in Jordan.
Seroprevalence and Risk Factors Materials and methods Study animal and design Horse population of Jordan, as reported by the latest statistical data from Jordan Ministry of Agriculture, is estimated to be 2182 horses (Department of Statistics, 2007). Two hundred and fifty-three horses were randomly enrolled in the study. Simple randomization technique was applied with stratification to geographical location. Stables and horse holdings were selected based on data obtained from the Ministry of Agriculture. Owners were asked to select horses to be enrolled in the study before revealing the study objectives. The horses were clinically normal and distributed over all provinces of Jordan. Enrolled horses from the different provinces were grouped into five major regions according to the geographical proximity and climatic similarities. Those were (i) Irbid, Ajlun and Jerash, (ii) Amman and Madaba, (iii) Ma’an, Karak, Tafelah and Aqaba, (iv) Mafraq and Zarka and (v) Jordan Valley and Balqa. From each region, around 50 horses were sampled. Data collection For each horse, a form was completed and included information about location, age, sex, breed, use of horse, type of nutrition (grazing or not), time owned by the owner and herd size. The age was classified into three groups; 10 years. The time, during which the owner has had the horse for, has been classified into two groups; 1 year. The herd size was further grouped into three groups; 40 horses. Blood samples A venous blood sample was collected from the jugular vein of each horse. The blood was collected into a plain tube,
© 2014 Blackwell Verlag GmbH • Transboundary and Emerging Diseases. 61 (Suppl. 1) (2014) 1–6
S. M. Abutarbush and A. M. Al-Majali
West Nile Virus Infection in Horses in Jordan
which was centrifuged. After harvesting the serum, it was stored at 20°C until it is used for serology. Serology The serum samples of all horses were screened for WNV antibodies using a commercial competitive ELISA test (ID Screenâ West Nile Competition; ID Vet), according to the manufacturer’s instructions. This competitive ELISA detects antibodies directed against the envelop protein of the West Nile virus. Serum samples that reacted positive using the previous ELISA test were further tested using commercial IgMcapture ELISA test (ID Screenâ West Nile IgM Capture; ID Vet), according to the manufacturer’s instructions. This ELISA detects anti-envelope protein IgM antibodies for West Nile virus in horses. According to the manufacturer, the sensitivity and specificity for both ELISAs are 100%. Statistical analysis Initially, univariable analysis was carried out by chi-square analysis. Variable were further assessed using a multivariable logistic regression model. All variables with P < 0.20 (two-sided) in the univariable analysis were further tested by the model. Variables were excluded from the model using a backward-stepwise approach. Statistical analyses were performed using SPSS software, version 17 (SPSS Inc., Chicago, IL, USA). Results Using the competitive ELISA test, 63 horses (n = 63, 24.9%) of the 253 surveyed were seropositive to WNV. Of the 63 horses, none had IgM antibodies for WNV. The distribution of the positive horses in Jordan is shown in Fig. 1. The region with the highest prevalence was Jordan Valley and Balqa, followed by Mafraq and Zarka, Ma’an, Karak, Tafelah and Aqaba, Amman and Madaba, and Irbid, Ajlun and Jerash, respectively. The chi-square univariable analysis revealed five factors (herd size, location, sex, use of the horse and grazing) with P value
