Chronic Volar Dislocation of the Distal Radioulnar Joint Stabilised by a Partial Extensor Carpi Ulnaris Tendon Strip - A Case Report.

Volar dislocation of the distal radioulnar joint is a rare injury that is often missed at initial presentation. We report a 21-year-old male patient who presented 2 months after sustaining this injury. He was successfully managed by open reduction and reconstruction of the dorsal radioulnar ligament using a partial distally based extensor carpi ulnaris tendon strip. A literature review showed only a few reported cases with varied methods for management. The technique utilised is analysed in comparison to the others. Level of Evidence: Level V (Therapeutic).

Serological Evidence of Antibodies to Rift Valley Fever Virus in Wild and Domestic Animals in Bauchi State, Nigeria.

Rift Valley fever (RVF) is an arthropod-borne zoonotic disease responsible for severe outbreaks in livestock and humans with concomitant economic losses in many countries in sub-Saharan Africa. The study, therefore, investigated the seroprevalence of the Rift Valley fever virus (RVFV) among wild and domestic animals. Blood samples were collected between 2013 and 2015 from 106 wild animals, 300 cattle (Bos indicus), and 200 horses (Equus caballus), respectively, in Yankari Game Reserve (YGR) and Sumu Wildlife Park (SWP) in Bauchi state, Nigeria. Harvested sera from blood were evaluated for the presence of anti-RVFV IgM/IgG antibodies. The overall seroprevalence in cattle was 11.3% (p = 0.677; 95% CI: 0.624-0.730) and in wildlife was 8.5% (p = 0.006; 95% CI: 0.00-0.60). The diversity of wildlife species sampled indicated seropositivity of 36.0% in waterbuck (Kobus ellipsiprymus), 25.0% in elephant (Loxodonta africana), 12.5% in eland (Taurotragus oryx), and 8.3% in wildebeest (Connochaetes taurinus). Whereas, samples from zebra (Equus quagga crawshayi), kudu (Tragelaphus strepsiceros), and hartebeest (Alcelaphus buselaphus caama) did not show detectable antibodies to RVFV, and seroprevalence in female (15.0%) wildlife species was higher than in males (4.5%) (p = 0.061). Classification of cattle into breed and sex showed no significant difference in seropositivity. Seropositivity of 12.0% was observed in White Fulani, 12.1% in Red Bororo, and 7.8% in Sokoto Gudali breeds of cattle (p = 0.677). Whereas, seropositivity of 13.6% was observed in females and 6.4% observed in males (p = 0.068). This study indicated the presence of antibodies to RVFV among some wild animals and cattle in the absence of a reported outbreak in the study area. The circulation of RVFV in the study area may pose a significant health risk to livestock, wildlife, and humans. Therefore, surveillance for RVFV should be intensified targeting mosquito vectors and humans in Bauchi state, Nigeria.

Toxicological evaluation of repeated administration of povidone iodine in cockerels.

Povidone-iodine (Polidine®) is a synthetic broad-spectrum antiseptic being applied topically to treat wounds and prevent their infection. It has been however reported with the assertions that it is effective in the treatment of infectious bursa disease (IBD) when administered orally by practicing Veterinarians and other poultry handlers. Acute kidney injury has been reported also with povidone iodine ingestion. Hence, in this study, graded dose administration was conducted to ascertain its safety profile. Forty chicks were obtained from a poultry hatchery in Ibadan, Oyo State. They were randomly divided into four (4) groups of ten chicks each. Group I served as negative control, groups II, III and IV were administered Polidine® at 1 mL/50 L, 1 mL/25 L, and 1 mL/10 L of water respectively for 7 days. Blood samples were collected on Days 3 and 7 post administration for determination of haematological and biochemical parameters. Liver and Kidney tissues were harvested following termination of the experiment and processed for histopathological examination. Results revealed no significant (p > 0.05) effect in the haematological and biochemical parameters of cockerels treated with Povidone iodine at 1 mL/50 and 25 L of water. On histopathological examination no lesion was also observed in the liver and kidney tissues of groups I, II and III (normal control, 1 mL/50 and 25 L respectively) when compared to group IV (1 mL PI /50 L of water) where lesions were recorded. Hence, this study has shown the relative safety of povidone iodine at different doses in cockerels.

Research Note: Evaluation of acute oral toxicity of povidone-iodine in cockerels using the up-and-down procedure.

Povidone-iodine (Polidine) is a synthetic broad-spectrum antiseptic and being applied topically to treat wounds and prevent their infection. It is however used by poultry farmers, field veterinarians, and other animal health workers with the claim that it is effective for treatment of infectious bursal disease when administered orally. Hence, an acute oral toxicity study was conducted to ascertain its safety profile. Ten cockerel chicks were randomly selected and divided into 2 groups of 5 chicks per group. One group served as the negative control, whereas the other group was administered povidone-iodine at a dose of 2,000 mg/kg of BW orally. The blood sample was collected at the end of the study to determine changes in hematological and biochemical parameters. In addition, vital organs were also harvested and preserved for histopathological examinations. The result showed that the median lethal dose (LD50) of the povidone-iodine is higher than 2,000 mg/kg of BW in cockerels. There were no significant changes in the hematological parameters measured. Biochemical evaluation (renal and liver function test) showed an increase in aspartate aminotransferase, alanine aminotransferase, and alkaline phosphatase levels after administration of povidone-iodine. The study indicated that the LD50 of povidone-iodine is higher than 2,000 mg/kg of BW of cockerels, and there were increases in urinary and liver enzymes at this dose.

Seroprevalence of Foot and Mouth Disease Virus Infection in Some Wildlife and Cattle in Bauchi State, Nigeria.

Foot and mouth disease (FMD) is an important transboundary viral disease of both domestic and wild cloven-hoofed animals characterized by high morbidity with devastating consequence on the livestock worldwide. Despite the endemic nature of FMD in Nigeria, little is known about the epidemiology of the disease at the wildlife-livestock interface level. To address this gap, blood samples were collected between 2013 and 2015 from some wildlife and cattle, respectively, within and around the Yankari Game Reserve and Sumu Wildlife Park in Bauchi State, Nigeria. Wild animals were immobilized using a combination of etorphine hydrochloride (M99® Krüger-Med South Africa) at 0.5-2 mg/kg and azaperone (Stresnil®, Janssen Pharmaceuticals (Pty.) Ltd., South Africa) at 0.1 mg/kg using a Dan-Inject® rifle (Dan-Inject APS, Sellerup Skovvej, Denmark) fitted with a 3 ml dart syringe and for reversal, naltrexone (Trexonil® Kruger-Med South Africa) at 1.5 mg IM was used, and cattle were restrained by the owners for blood collection. Harvested sera from blood were screened for presence of antibodies against the foot and mouth disease virus (FMDV) using the PrioCHECK® 3ABC NSP ELISA kit, and positive samples were serotyped using solid-phase competitive ELISA, (IZSLER Brescia, Italy). Out of the 353 sera collected from cattle and wildlife 197 (65.7%) and 13 (24.5%) (P < 0.05), respectively, tested positive for antibodies to the highly conserved nonstructural 3ABC protein of FMDV by the FMDV-NS blocking ELISA. Classification of cattle into breed and sex showed that detectable antibodies to FMDV were higher (P < 0.05) in White Fulani 157 (72.8%) than in Red Bororo 23 (39.7%) and Sokoto Gudali 17 (33.3%) breeds of cattle, whereas in females, detectable FMDV antibodies were higher (P < 0.05) 150 (72.8%) than in males 47 (50.0%). In the wildlife species, antibodies to FMDV were detected in the waterbucks 2 (28.6%), elephant 1 (25.0%), wildebeests 4 (33.3%), and elands 6 (25.0%). Four serotypes of FMDV: O, A, SAT 1, and SAT 2 were detected from the 3ABC positive reactors in waterbucks, elephants, wildebeests, and elands. The results showed presence of antibodies to FMDV in some wildlife and cattle and suggested that wildlife could equally play an important role in the overall epidemiology of FMD in Nigeria. FMD surveillance system, control, and prevention program should be intensified in the study area.

Application of loop-mediated isothermal amplification assay in the detection of herpesvirus of turkey (FC 126 strain) from chicken samples in Nigeria.

AIM: This study was designed to optimize and apply the use of loop-mediated isothermal amplification (LAMP) as an alternative to conventional polymerase chain reaction (PCR) for the detection of herpesvirus of turkeys (HVT) (FC 126 strain) in vaccinated and non-vaccinated poultry in Nigeria. MATERIALS AND METHODS: HVT positive control (vaccine) was used for optimization of LAMP using six primers that target the HVT070 gene sequence of the virus. These primers can differentiate HVT, a Marek's disease virus (MDV) serotype 3 from MDV serotypes 1 and 2. Samples were collected from clinical cases of Marek's disease (MD) in chickens, processed and subjected to LAMP and PCR. RESULTS: LAMP assay for HVT was optimized. HVT was detected in 60% (3/5) and 100% (5/5) of the samples analyzed by PCR and LAMP, respectively. HVT was detected in the feathers, liver, skin, and spleen with average DNA purity of 3.05-4.52 µg DNA/mg (A260/A280) using LAMP. Conventional PCR detected HVT in two vaccinated and one unvaccinated chicken samples, while LAMP detected HVT in two vaccinated and three unvaccinated corresponding chicken samples. However, LAMP was a faster and simpler technique to carry out than PCR. CONCLUSION: LAMP assay for the detection of HVT was optimized. LAMP and PCR detected HVT in clinical samples collected. LAMP assay can be a very good alternative to PCR for detection of HVT and other viruses. This is the first report of the use of LAMP for the detection of viruses of veterinary importance in Nigeria. LAMP should be optimized as a diagnostic and research tool for investigation of poultry diseases such as MD in Nigeria.

Transmission rate and reproductive number of the H5N1 highly pathogenic avian influenza virus during the December 2005-July 2008 epidemic in Nigeria.

We quantified the between-village transmission rate, ß (the rate of transmission of H5N1 HPAI virus per effective contact), and the reproductive number, Re (the average number of outbreaks caused by one infectious village during its entire infectious period), of H5N1 highly pathogenic avian influenza (HPAI) virus in Nigeria using outbreak data collected between December 2005 and July 2008. We classified the outbreaks into two phases to assess the effectiveness of the control measures implemented. Phase 1 (December 2005-October 2006) represents the period when the Federal Government of Nigeria managed the HPAI surveillance and response measures, while Phase 2 (November 2006-July 2008) represents the time during which the Nigeria Avian Influenza Control and Human Pandemic Preparedness project (NAICP), funded by a World Bank credit of US$ 50 million, had taken over the management of most of the interventions. We used a total of 204 outbreaks from 176 villages that occurred in 78 local government areas of 25 states. The compartmental susceptible-infectious model was used as the analytical tool. Means and 95% percentile confidence intervals were obtained using bootstrapping techniques. The overall mean ß (assuming a duration of infectiousness, T, of 12 days) was 0.07/day (95% percentile confidence interval: 0.06-0.09). The first and second phases of the epidemic had comparable ß estimates of 0.06/day (0.04-0.09) and 0.08/day (0.06-0.10), respectively. The Re of the virus associated with these ß and T estimates was 0.9 (0.7-1.1); the first and second phases of the epidemic had Re of 0.84 (0.5-1.2) and 0.9 (0.6-1.2), respectively. We conclude that the intervention measures implemented in the second phase of the epidemic had comparable effects to those implemented during the first phase and that the Re of the epidemic was low, indicating that the Nigeria H5N1 HPAI epidemic was unstable.

Characterization of field and vaccine infectious bursal disease viruses from Nigeria revealing possible virulence and regional markers in the VP2 minor hydrophilic peaks.

Outbreaks of infectious bursal disease in vaccinated chicken flocks are frequent in Nigeria. For the control of infectious bursal disease, live vaccines based on foreign infectious bursal disease virus (IBDV) strains are used. The present study investigated the phylogenetic relationship between field and vaccine IBDV strains from northwestern Nigeria. Thirty field IBDV strains and three commercial vaccines strains were characterized through sequencing the VP2 hypervariable region. In addition, the complete genome segment A coding region for two vaccines and two field strains was sequenced. The deduced amino acid sequences (position 212 to 331) of IBDV strains from Nigeria and other regions of the world were aligned and possible regional and virulence markers were identified associated with VP2 minor hydrophilic peaks. Reversion to virulence of a vaccine strain with a Q to L mutation at position 253 was observed. Phylogenetic analyses revealed a unique cluster of northwest Nigerian field IBDV strains alone or related to imported characterized classical and very virulent IBDV vaccines. The results suggest that when IBDV strains spread from their region of origin to a different region they mutate alongside indigenous field strains but may retain their identity on the VP2 region.

Identification of potential risk factors associated with highly pathogenic avian influenza subtype H5N1 outbreak occurrence in Lagos and Kano States, Nigeria, during the 2006-2007 epidemics.

Highly pathogenic avian influenza HPAI H5N1 was first reported in Africa in 2006, in Nigeria. The country experienced severe outbreaks in 2006 and 2007, strongly affecting the poultry population. Current knowledge on potential risk factors for HPAI H5N1 occurrence in poultry farms in Nigeria is limited. Therefore, we conducted a case-control study to identify potential farm-level risk factors for HPAI H5N1 occurrence in two areas of the country that were affected by the disease in 2006 and 2007, namely the States of Lagos and Kano. A case-control study was conducted at the farm level. A convenience sample of 110 farms was surveyed. Data on farm characteristics, farm management and trade practices were collected. Logistic regression was used to identify factors associated with farms that confirmed positive for HPAI. Having a neighbouring poultry farm was identified as a potential risk factor for disease occurrence [OR, 5.23; 95% CI, (0.88-30.97); P-value = 0.048]. Farm staff washing their hands before handling birds was a protective factor [OR, 0.14; 95% CI, (0.05-0.37); P-value <0.001], as well as not allowing traders to enter the farm [OR, 0.23; 95% CI, (0.08-0.70); P-value = 0.008]. Our study highlighted the importance of trade and proximity between poultry farms in the epidemiology of HPAI H5N1 and the role of biosecurity in disease prevention in Kano and Lagos States. Despite the limitations owing to the sampling strategy, these results are consistent with other risk factor studies previously conducted on HPAI H5N1 in both Africa and other regions, suggesting similar risk factor patterns for HPAI H5N1 virus spread and substantiating current knowledge regarding the epidemiology of the disease. Finally, this study generated information from areas where data are difficult to obtain.

Incidence of highly pathogenic avian influenza H5N1 in Nigeria, 2005-2008.

Outbreaks of highly pathogenic avian influenza (HPAI) H5N1 occurred in Nigeria between December 2005 and July 2008. We describe temporal and spatial characteristics of these outbreaks at State and Local Government Area (LGA) levels. A total of 25 of 37 States (67.6%; Exact 95% CI: 50.2-82.0%) and 81 of 774 LGAs (10.5%; Exact 95% CI: 8.4-12.8%) were affected by HPAI outbreaks over the period from 2005 to 2008. The incidence risk of HPAI outbreak occurrence at the State level was 5.6% (0.7-18.7%) for 2005, 50.0% (30.7-69.4%) for 2006, 54.5% (29.9-80.3%) for 2007 and 0% for 2008. Only very few LGAs experienced HPAI outbreaks within the affected States. The incidence risk of HPAI outbreak occurrence on a LGA level was 0.3% (0.0-0.9%) for 2005, 6.6% (4.9-8.6%) for 2006, 4.2% (2.9-6.0%) for 2007 and 0% for 2008. The mean period between farmers noticing HPAI outbreaks and reporting them to veterinary authorities, and between reporting HPAI outbreaks and the depopulation of infected premises, was for both 4.5 days; both periods also had medians of 1 day. We have estimated the spatially smoothed incidence risk for the whole outbreak period and identified the existence of a large corridor in the western part of Nigeria and a smaller corridor in south-eastern part, where the risk of HPAI occurrence was lower than in the rest of the country. The effect of HPAI control policies on the outbreaks patterns are discussed, as well as possible reasons why HPAI did not become endemic in Nigeria.

Serologic evidence of infection with H5 subtype influenza virus in apparently healthy local chickens in Kaduna State, Nigeria.

A serologic survey for antibodies against H5 subtype influenza virus in 605 apparently healthy local chickens using a hemagglutination inhibition test was carried out in 12 local government areas of Kaduna state, Nigeria. An overall prevalence of 18.1% was recorded, with a higher prevalence of 27.3% in six local government areas that have not reported outbreaks of highly pathogenic avian influenza (HPAI) H5N1 virus and a lower prevalence of 7.5% in six local government areas that had reported and confirmed outbreaks of HPAI H5N1 virus between 2006 and 2007. There was association between the presence of ducks and detection of H5 antibodies (P = 0.000, odds ratio = 0.22). The implication of this finding is discussed, although a virologic investigation to verify the findings of this study is highly recommended.

Changes in weight gain, faecal oocyst count and packed cell volume of Eimeria tenella-infected broilers treated with a wild mushroom (Ganoderma lucidum) aqueous extract.

An experiment was conducted to study the effect of a wild Ganoderma lucidum aqueous extract in coccidian-infected broilers. At 6 weeks of age the birds were randomly allocated to 6 treatment groups of 20 Ross broilers each in wire cages. Groups A, B and C were infected with Eimeria tenella Houghton strain at the rate of 36 250 sporulated oocysts/ml per bird. The remaining 3 groups D, E and F were uninfected controls. At 7 weeks the birds in group A were treated with G. lucidum aqueous extract and those in B with amprolium in drinking water ad libitum at the rate of 200 mg/ml each for 7 days consecutively. Body weight gain, feed intake, faecal oocyst output and some haematological parameters were monitored. The result showed that all the infected birds in groups A, B and C had clinical signs of weakness and reduced appetite on day 4 post-infection. By the 5th day post-infection their faeces became bloody and watery, and large numbers of E. tenella oocysts were present in the faeces. On day 3 after treatment the oocysts detected were considerably reduced in both treated groups A and B and slightly higher in the untreated group C. The faeces of the uninfected control groups were normal and free of coccidial oocysts. After treatment for 7 days no coccidial oocysts were found in faeces of the birds that had been treated. Infected, untreated birds showed a slight drop in feed intake and weight gain from 7 to 8 weeks of age. The final mean weight gain recorded in the treated groups A and B was comparable to that of the uninfected birds in the 3 control groups, while it was lower in the untreated group C. The feed to gain ratio was higher in C than in the other groups. A slight drop in packed cell volume was observed in groups A, B and C at 7 weeks of age, 1 week after infection. This study showed that treatment with G. lucidum results in a marked reduction in the number of E. tenella oocysts shed in the faeces, leading to improved weight gain and decreased weight loss. The results confirmed the virulence of the Houghton strain of E. tenella and the effectiveness of both amprolium and G. lucidum extract against E. tenella.

Haemagglutination inhibition antibodies, rectal temperature and total protein of chickens infected with a local Nigerian isolate of velogenic Newcastle disease virus.

Changes in values of haemagglutination inhibition (HI) antibody titre, rectal temperature (RT) and total protein (TP) were determined for Shaver Brown chickens infected with Newcastle disease virus (NDV) Kudu 113. The infected chickens came down with Newcastle disease by day 3 post infection (PI). The major clinical signs were depression, greenish diarrhoea, paralysis of legs and wings, opisthotonus and torticolis. Mortality and morbidity were 52% and 1000%, respectively. There were haemorrhagic lesions in the wall of the intestine, proventricular mucosa and caecal tonsils. There were necrosis and mononuclear cell infiltration of the liver, kidney and spleen. There was a significant increase in daily mean HI antibody titres from days 3 to 9 PI. Similarly, significant rise in daily mean RTs were noticed in the infected chickens from days 1 to 13 PI. On the other hand, there was a decrease in daily mean TP concentrations of infected chickens, beginning from day 3 PI, and the lowest concentration of 2.60 +/- 0.15 g/dl was obtained by days 7 and 11 PI. The values of HI, RT and TP for the control chickens were relatively constant during the experiment. The correlation coefficient (r) between HI and RT was positive and highly significant (r = 0.725, p<0.001), while the relationship between HI and TP was negative but highly significant (r = -0.712, p<0.001). It was concluded that NDV Kudu 113 induced increases in values of HI and RT, which occurred concurrently with a decrease in TP concentrations of infected chickens.

In vitro screening of two Nigerian medicinal plants (Vernonia amygdalina and Annona senegalensis) for anthelmintic activity.

Vernonia amygdalina and Annona senegalensis, two plants used by local/smallholder livestock farmers in Nigeria as anthelmintic were screened for in vitro anthelmintic activity using Haemonchus contortus eggs. The extract of V. amygdalina did not show any significant activity at concentrations up to 11.2mg/ml. The extract of A. senegalensis showed significant (P<0.001) reduction in egg hatch at a concentration of 7.1mg/ml. The in vitro fecal culture of eggs with A. senegalensis showed significant (P<0.001) reductions in larval recovery with increasing concentrations from 1 to 10% ((w/w) in fecal culture) when whole ground plant material was used. Although traditional veterinary healers and local livestock farmers claim both plants are effective as anthelmintics, our result indicate that only A. senegalensis showed promising anthelmintic activity especially with use of ground whole plant materials as used by some farmers.

Effects of baicalein on prostanoid generation from the lung and contractile responses of the trachea in guinea pig.

Effects of baicalein on release of slow reacting substance of anaphylaxis (SRS-A) or leukotriene (LT) from the sensitized guinea pig lung after antigen challenge and tonus of guinea pig tracheal muscles were studied. Baicalein inhibited release of SRS-A from sensitized guinea pig lung after antigen challenge. High-performance liquid chromatography (HPLC) analysis revealed that released SRS-A consisted to LTC4 and D4. Baicalein also reduced release of LTC4 and D4 from the sensitized lung after antigen challenge. Baicalein relaxed the isolated guinea pig tracheal smooth muscle contracted by LTD4, carbachol or histamine. However, this compound produced a contraction when the tracheal muscle was contracted by prostaglandin F2 alpha(PGF2 alpha). This contraction by baicalein was abolished by pretreatment with indomethacin, a cyclooxygenase inhibitor. Baicalein elicited a relaxation in the normal non-sensitized preparation but a contraction in the tissue isolated from actively sensitized guinea pig in 4 among 7 cases. Baicalein also produced a contraction in the trachea pretreated with phorbol dibutyrate and contracted by carbachol, which was eliminated after treatment with indomethacin. The results suggest that baicalein exerts action via, at least, two different mechanisms, the inhibition of releasing SRS-A (LTs) and direct relaxing effects on the trachea. Besides, baicalein seems to produce contraction under certain conditions, which may involve stimulation of the cyclooxygenase pathway.

Snakebites in poultry.

Eight cases of snakebite involving 5 flocks were seen in chickens, ducks and turkeys in the Avian Ambulatory Clinic of the Veterinary Teaching Hospital, Zaria, between July 1983 and August 1990. Most of the clients resided in areas with large canopies of trees and bushes. There was a large gully just behind the residential areas. Twenty-one birds were bitten; chickens constituted 85.7%, turkeys 12% and ducks 4.8% of this number. Hens incubating eggs were most vulnerable. Cocks were usually bitten when they attacked the snakes. Clinical signs were similar to those reported in other animal species. Gross lesions included congestion of visceral organs and hemorrhage in the viscera, around the bite sites and in the sc tissues. There was also im edema around bite sites. The fatality rate was 87.5%.

Campylobacter fetus subsp. jejuni in poultry reared under different management systems in Nigeria.

Cloacal swabs from 487 live birds in 36 flocks and 70 poultry carcasses were cultured for Campylobacter fetus subsp. jejuni. It was isolated from 12.3% of the birds in 19 flocks. Chickens, turkeys, and guinea fowl differed from one another in isolation rates of the organism. Management system affected its occurrence, and only 7.1% of eviscerated carcasses yielded it. It was concluded that bird species, management system, and immersing slaughtered poultry in boiling water before dressing affect recovery of C. fetus subsp. jejuni from live birds and carcasses.

Challenge study on infectious bursal disease in chicks derived from vaccinated hens.

Presence and levels of maternal antibody (MA) in broiler chicks derived from hens vaccinated with a live infectious bursal disease (IBD) vaccine were investigated by a quantitative agar-gel precipitin test. At day old 100% of the chicks tested had MA; by 17 days of age it was present in only 10%. The mean MA level at day old was 337.5 UK units/ml but decreased to 6.3 UK units/ml at 17 days of age. Randomly selected chicks from the pool studied were challenged at weekly intervals from day old for 29 days with an IBD virus obtained from a natural outbreak. Subclinical and clinical disease were observed in chicks challenged at eight and 29 days of age respectively.

Infectious bursal disease and spirochetosis in pullet chicks.

Infectious bursal disease and spirochetosis were diagnosed simultaneously in a flock of 100 six-week-old babcock pullet chicks. Larvae of the fowl tick Argas persicus were found on the bodies of the chicks. Thirty-nine of the chicks died 5 days after the onset of clinical signs.