Serological and molecular detection of bovine brucellosis at institutional livestock farms in Punjab, Pakistan

Bovine brucellosis remains a persistent infection in ruminants in Pakistan. A total of 828 (409 buffaloes and 419 cattle) sera were collected from 11 institutional-owned livestock farms in Punjab, Pakistan. The samples were tested by rose bengal plate agglutination test (RBPT) and indirect enzyme-linked immunosorbent assay (iELISA). The seroprevalence along with 95% confidence interval (CI) was determined. Univariable and multivariable analysis of the epidemiological background data was conducted and odds ratio (OR) was calculated to understand any association between the risk factors and the seroprevalence. An overall seroprevalence of 3.9% (Positive/Tested = 32/828) and 3.3% (27/828) was detected by RBPT and iELISA, respectively. The seroprevalence of 5.6% (CI 3.6–8.3) and 4.7%, (CI 2.8–7.2) and the odds ratio of 2.63 (CI 1.20–5.77) and 2.50 (CI 1.08–5.78) for testing positive by RBPT and iELISA, respectively were significantly higher (p < 0.05) in buffaloes than in cattle. Breed, sex, history of abortion and retention of fetal membranes (RFM) in the animals were not found statistically significantly associated with the infection. RBPT and iELISA based results agreed almost perfect (k = 0.877). In total, Brucella abortus-DNA (9/27) was amplified from seropositive samples by real-time polymerase chain reaction. This study identified for the first time the etiological agents of brucellosis at a molecular level at institutional-owned livestock farms in Pakistan. View Full-Tex

Revisiting Brucellosis in Small Ruminants of Western Border Areas in Pakistan

Brucellosis, globally known bacterial zoonosis, is endemic to Pakistan. B. abortus in bovines, B. melitensis in small ruminants and B. canis in dogs mainly cause this disease. A total of 1821 sera (1196 from sheep and 625 from goats) from animal herds near the Pakistan–Afghanistan border were collected. In parallel testing of sera for anti-Brucella antibodies (B. abortus and B. melitensis) was carried out by RBPT and indirect ELISA. The presence of Brucella DNA in sera was tested by real-time PCR. The overall percentage of seropositive samples was 0.99 (18/1821) by both tests. All positive samples originated from Baluchistan territory which translated into 1.76% (18/1021). None of the positive sera had signals for Brucella DNA and none of sera from goats carried detectable antibodies. Both tests showed an almost perfect agreement with Kappa statistics. The flock size was found to be associated with the presence of anti-Brucella antibodies. The samples of Khyber Pakhtunkhwa (KPK) tested negative in both serological tests and hence were not processed for real-time PCR. The present study shows the presence of anti-Brucella antibodies in sheep in the Baluchistan region of Pakistan. Diagnostic services need to be improved and test and slaughter policies might be implemented for eradication of Brucella infection in these areas. Awareness about the infection is needed at the farmer’s level. Isolation and molecular biology of the isolates could help with understanding the prevailing etiology in a better way

Epidemiologie des hämorrhagischen Krim-Kongo-Fiebervirus bei Zecken und Nutztieren in Belutschistan, Pakistan

Crimean-Congo haemorrhagic fever (CCHF) is a tick-borne zoonotic disease caused by the arbovirus Crimean-Congo haemorrhagic fever virus (CCHFV). It causes fatal haemorrhagic disease in human. Ticks considered as reservoir and vector for CCHFV. Livestock serve as a transient reservoir for this virus, but do not show clinical signs. In part I (3.2) of this thesis, a cross-sectional study has been conducted from July to September 2016, in which sheep and goats in Balochistan, Pakistan, were examined to determine the CCHFV seroprevalence, spatial distribution of seropositive sheep and goats, and to identify potential risk factors for seropositivity to CCHFV in these animals. To this end, farms and animals selected by systematic sampling, blood samples from 800 sheep and 800 goats collected and information regarding farm management and the kept animals were retrieved using a standard questionnaire. Sera tested for antibodies against CCHFV in two independent ELISA formats and an immunofluorescence assay (IFA) following a hierarchical diagnostic decision tree. By these assays 149 (19%, 95%-CI: 16%-21%) out of 800 sheep serum samples and 37 (5%, 95%-CI: 3%-6%) out of 800 goat serum samples were positive for CCHFV-specific IgG antibodies. Interestingly, at least 8 (5%, 95%-CI: 2%-10%) out of 160 sera pools were from CCHFV viremic sheep, as sera (in pools of 5) tested positive for CCHFV genome by real time PCR (RT-qPCR). Risk factor analysis revealed that the open type of housing (OR=3.76, 95%-CI:1.57-9.56, pvalue=0.003), grazing (OR=4.18, 95%-CI:1.79-10.37, p-value=0.001), presence of vegetation in or around the farm (OR= 3.13, 95%-CI: 1.07-10.15, p-value=0.043), lack of treatment against ticks (OR=3.31, 95%-CI: 1.16-10.21, p-value=0.029), absence of rural poultry (OR=2.93, 95%-CI: 1.41-6.29, p-value=0.004), animals with age > 2 years (OR=4.15, 95%-CI: 2.84-6.19, pvalue<0.001), animals infested with ticks (OR=2.35, 95%-CI: 1.59-3.52, p-value<0.001), and sheep species (OR=4.72, 95%-CI:3.24-6.86, p-value<0.001) represented statistically significant risk factors associated with seropositivity to CCHFV. Taken together this part of study confirms the circulation of CCHFV in livestock in Balochistan, Pakistan. The identification of risk factors might help to reduce the risk of infection in sheep and goats, which may also mitigate the risk for human infection. An interesting option for reducing the risk of CCHFV infection in small ruminants is keeping also chickens, since they pick ticks that transmit CCHFV. In part II (3.3), a cross-sectional study has been conducted from September to November 2017, in the province of Balochistan, Pakistan. Ticks were collected from cattle, sheep and goats in the livestock farms. The ticks were identified morphologically and the result confirmed by genotyping. Further, ticks were analysed to detect CCHFV genome by one-step multiplex real-time RT-qPCR, and positive ticks were sequenced to determine the CCHFV genotype. In 529 livestock infested ticks, 525 (99%) ticks belonged to the genus Hyalomma, and four (1%) ticks were from the genus Rhipicephalus. Within the genus Hyalomma, H. marginatum (n=149; male=92, female=57), H. excavatum (n=135; male=96, female=39), H. dromedarii (n=117; male=101, female=16), H. anatolicum (n=82; male=61, female=21), and H. scupense (n=42; male= 36, female=6) were identified. In the genus Rhipicephalus, R. microplus (n=3), and R. turanicus (n=1) were found. The tick infestations on ruminants were 58 % in sheep (n=307), 28 % in goats (n=146), and 14 % in cattle (n=76). All collected ticks were adults. Four percent (20 out of 525, 95%-CI: 2%-6%) ticks were positive for CCHFV genome (S segment). All CCHFV sequences obtained from the ticks clustered in the Asia-1 genotype. Among the CCHFV-positive ticks, 75% (15 out of 20) were female and 25% (5 out of 20) were male. CCHFV genome was detected most frequently in H. marginatum (30%, 6 out of 20), followed by, H. dromedarii (25%, 5 out of 20), H. excavatum (20%, 4 out of 20), H. anatolicum (20%, 4 out of 20), and H. scupense (5%, 1 out of 20). All CCHFVpositive ticks were found on sheep. The highest number of CCHFV-positive ticks was detected in the Kalat district (60%, 12 out of 20), followed by Quetta (30%, 6 out of 20) and Killa Abdullah (10%, 2 out of 20) districts. This part of the study confirms the circulation of CCHFV in ticks in the south-western part (Balochistan) of Pakistan. It is imperative to take effective tick-control measures in this area, especially to control livestock infestation with ticks, to reduce the risk of CCHF outbreaks in the human population.Krim-Kongo-Hämorrhagisches Fieber (CCHF) ist eine durch Zecken übertragene Zoonose, die durch das Krim-Kongo-Hämorrhagisches Fieber Virus (CCHFV), ein Arbovirus, verursacht wird. Es verursacht tödliche hämorrhagische Erkrankungen beim Menschen. Zecken gelten als Reservoir und Vektor für CCHFV. Vieh dient als vorübergehendes Reservoir für dieses Virus, zeigt jedoch keine klinischen Anzeichen. In Teil I (3.2) wurde von Juli bis September 2016 eine Querschnittsstudie durchgeführt, in der Schafe und Ziegen in Belutschistan, Pakistan, untersucht wurden, um die CCHFV-Seroprävalenz, die räumliche Verteilung von seropositiven Schafen und Ziegen sowie mögliche Risikofaktoren für Seropositivität gegenüber CCHFV bei diesen Tieren zu bestimmen. Zu diesem Zweck wurden Betriebe und Tiere durch systematische Probenahme ausgewählt, Blutproben von 800 Schafen und 800 Ziegen entnommen und Informationen zur Betriebsführung und den gehaltenen Tieren unter Verwendung eines Standardfragebogens abgerufen. Die Seren wurden in zwei unabhängigen ELISA-Formaten und einem Immunfluoreszenz-Assay (IFA) nach einem hierarchischen diagnostischen Entscheidungsbaum auf Antikörper gegen CCHFV getestet. Mit diesen Tests waren 149 (19%, 95%-CI: 16%-21%) von 800 Schafserumproben und 37 (5%, 95%-CI: 3%-6%) von 800 Ziegenserumproben positiv für CCHFV-spezifische IgG-Antikörper. Interessanterweise stammten mindestens 8 (5%, 95%-CI: 2%-10%) von 160 Serumpools von virämischen CCHFV-Schafen, da Seren (in Pools von 5) in der realtime-PCR positiv auf das CCHFV-Genom getestet wurden. Die Risikofaktoranalyse ergab, dass Offenställe (OR = 3,76, 95%-CI: 1,57-9,56, p-Wert = 0,003), Weidegang (OR = 4,18, 95%-CI: 1,79-10,37, p-Wert = 0,001), Vorhandensein von Vegetation in oder um den Betrieb (OR = 3,13, 95%-CI: 1,07-10,15, p-Wert = 0,043), fehlende Behandlung gegen Zecken (OR = 3,31, 95%-CI: 1,16-10,21, p-Wert = 0,029), Abwesenheit von Geflügel (OR = 2,93, 95%-CI: 1,41-6,29, p-Wert = 0,004), Tiere mit einem Alter von > 2 Jahren (OR = 4,15, 95%-CI: 2,84-6,19, p-Wert <0,001), mit Zecken befallene Tiere (OR = 2,35, 95%-CI: 1,59-3,52, p-Wert <0,001) und Schafarten (OR = 4,72, 95%-CI: 3,24-6,86, p-Wert <0,001) statistisch signifikante Risikofaktoren darstellten, die mit der Seropositivität gegenüber CCHFV assoziiert waren. Somit bestätigt dieser Teil der Studie die Verbreitung von CCHFV bei Nutztieren in Belutschistan, Pakistan. Die Identifizierung von Risikofaktoren könnte dazu beitragen, das Infektionsrisiko bei Schafen und Ziegen zu verringern, was auch das Risiko einer Infektion des Menschen verringern kann. Eine interessante Option zur Verringerung des Risikos einer CCHFV-Infektion bei kleinen Wiederkäuern ist die Haltung von Hühnern, da sie Zeckenfressen, die CCHFV übertragen. In Teil II (3.3) wurde von September bis November 2017 eine Querschnittsstudie in der pakistanischen Provinz Belutschistan durchgeführt. In den Viehbetrieben wurden Zecken von Rindern, Schafen und Ziegen gesammelt. Die Zecken wurden morphologisch identifiziert und der Befund durch Genom-Typisierung bestätigt. Weiterhin wurden Zecken analysiert, um das CCHFV-Genom durch Multiplex-realtime-RT-qPCR nachzuweisen. Positive Proben wurden sequenziert, um den CCHFV-Genotyp zu bestimmen. Von insgesamt 529 Zecken, die bei Schafen, Ziegen oder Rindern abgesammelt wurden, gehörten 525 (99%) zur Gattung Hyalomma, und 4 (1%) zur Gattung Rhipicephalus 4 (1%). In der Gattung Hyalomma waren H. marginatum (n = 149; männlich = 92, weiblich = 57), H. excavatum (n = 135; männlich = 96, weiblich = 39), H. dromedarii (n = 117; männlich = 101) weiblich = 16), H. anatolicum (n = 82; männlich = 61, weiblich = 21) und H. scupense (n = 42; männlich = 36, weiblich = 6). In der Gattung Rhipicephalus wurden R. microplus (n = 3) und R. turanicus (n = 1) identifiziert. Insgesamt waren 307 Schafe (58%) 146 Ziegen (28%) und 76 Rinder (14%) mit den Zecken befallen. Alle abgesammelten Zecken waren Adulte. 4% (20 von 525, 95%-CI: 2%-6%) Zecken waren positiv für das Genom des CCHFV-S-Segments. Alle CCHFV-Sequenzen aus den Zecken clusterten im Asia-1-Genotyp. Unter den CCHFV-positiven Zecken waren 75% (15 von 20) weiblich und 25% (5 von 20) männlich. Das CCHFV-Genom wurde in H. marginatum (30%, 6 von 20) am häufigsten nachgewiesen, gefolgt von H. dromedarii (25%, 5 von 20), H. excavatum (20%, 4 von 20), H. anatolicum (20%, 4 von 20) und H. scupense (5%, 1 von 20). Alle CCHFV-positiven Zecken wurden an Schafen gefunden. Die höchste Anzahl von CCHFV-positiven Zecken war im Bezirk Kalat zu verzeichnen (60%, 12 von 20), gefolgt von den Bezirken Quetta (30%, 6 von 20) und Killa Abdullah (10%, 2 von 20). Dieser Teil der Studie bestätigt die Verbreitung von CCHFV in Zecken im südwestlichen Teil Pakistans (Belutschistan). In diesem Bereich sollten dringend wirksame Maßnahmen zur Zeckenbekämpfung ergriffen werden, insbesondere um den Zeckenbefall bei Nutztieren zu bekämpfen, und so das Risiko von Ausbrüchen von CCHF in der menschlichen Bevölkerung zu verringern

Ticks on the run: A mathematical model of Crimean-Congo Haemorrhagic Fever (CCHF)-key factors for transmission

Crimean-Congo haemorrhagic fever (CCHF) is a tick-borne zoonotic disease caused by the Crimean-Congo hemorrhagic fever virus (CCHFV). Ticks belonging to the genus \textit{Hyalomma} are the main vectors and reservoir for the virus. It is maintained in nature in an endemic vertebrate-tick-vertebrate cycle. CCHFV is prevalent in wide geographical areas including Asia, Africa, South-Eastern Europe and the Middle East. Over the last decade, several outbreaks of CCHFV have been observed in Europe, mainly in Mediterranean countries. Due to the high case/fatality ratio of CCHFV in human sometimes, it is of great importance for public health. Climate change and the invasion of CCHFV vectors in Central Europe suggest that the establishment of the transmission in Central Europe may be possible in future. We developed a compartment-based nonlinear Ordinary Differential Equation (ODE) system to model the disease transmission cycle including blood sucking ticks, livestock and human. Sensitivity analysis of the basic reproduction number $R_0$ shows that decreasing in the tick survival time is an efficient method to eradicate the disease. The model supports us in understanding the influence of different model parameters on the spread of CCHFV. Tick to tick transmission through co-feeding and the CCHFV circulation through trasstadial and transovarial stages are important factors to sustain the disease cycle. The proposed model dynamics are calibrated through an empirical multi-country analysis and multidimensional scaling reveals the disease-parameter sets of different countries burdened with CCHF are different. This necessary information may help us to select most efficient control strategies

Serological and Molecular Detection of Bovine Brucellosis at Institutional Livestock Farms in Punjab, Pakistan

Bovine brucellosis remains a persistent infection in ruminants in Pakistan. A total of 828 (409 buffaloes and 419 cattle) sera were collected from 11 institutional-owned livestock farms in Punjab, Pakistan. The samples were tested by rose bengal plate agglutination test (RBPT) and indirect enzyme-linked immunosorbent assay (iELISA). The seroprevalence along with 95% confidence interval (CI) was determined. Univariable and multivariable analysis of the epidemiological background data was conducted and odds ratio (OR) was calculated to understand any association between the risk factors and the seroprevalence. An overall seroprevalence of 3.9% (Positive/Tested = 32/828) and 3.3% (27/828) was detected by RBPT and iELISA, respectively. The seroprevalence of 5.6% (CI 3.6&ndash;8.3) and 4.7%, (CI 2.8&ndash;7.2) and the odds ratio of 2.63 (CI 1.20&ndash;5.77) and 2.50 (CI 1.08&ndash;5.78) for testing positive by RBPT and iELISA, respectively were significantly higher (p &lt; 0.05) in buffaloes than in cattle. Breed, sex, history of abortion and retention of fetal membranes (RFM) in the animals were not found statistically significantly associated with the infection. RBPT and iELISA based results agreed almost perfect (k = 0.877). In total, Brucella abortus-DNA (9/27) was amplified from seropositive samples by real-time polymerase chain reaction. This study identified for the first time the etiological agents of brucellosis at a molecular level at institutional-owned livestock farms in Pakistan