Serological and Molecular Investigation of Brucella Species in Dogs in Pakistan

Brucellosis is an important bacterial zoonosis caused by B. abortus and B. melitensis in Pakistan. The status of canine brucellosis caused by B. canis remains obscure. In total, 181 serum samples were collected from stray and working dogs in two different prefectures viz. Faisalabad (n = 87) and Bahawalpur (n = 94). Presence of antibodies against B. canis and B. abortus/B. melitensis was determined using the slow agglutination test (SAT) and ELISA, respectively. Real-time PCR was performed to detect and differentiate Brucella DNA at the species level. In Faisalabad, the serological prevalence was found to be 9.2% (8/87) and 10.3% (9/87) by SAT and ELISA, respectively. Only one of the ELISA positive samples (1.15%) yielded amplification for B. abortus DNA. In Bahawalpur, 63.8% (60/94) samples were found positive by SAT; however, none of the samples was positive by ELISA or by real-time PCR. Location, age (≥1 year) and body condition (weak) were found to be associated with B. canis infection, whereas presence of wounds was found to be associated with B. abortus infection only. These findings point towards a risk of transmission from dog to livestock and humans and vice versa. The study expects to draw the attention of concerned authorities towards infection prevention and animal welfare. This study warrants further epidemiological investigation on brucellosis in pet dogs and their owners. To the best of our knowledge, this is first ever report on B. canis and B. abortus in dogs in Pakistan

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

Effectiveness of an antimicrobial treatment scheme in a confined glanders outbreak

BACKGROUND: Glanders is a contagious and fatal zoonotic disease of solipeds caused by the Gram-negative bacterium Burkholderia (B.) mallei. Although regulations call for culling of diseased animals, certain situations e.g. wild life conservation, highly valuable breeding stock, could benefit from effective treatment schemes and post-exposure prophylaxis. RESULTS: Twenty three culture positive glanderous horses were successfully treated during a confined outbreak by applying a treatment protocol of 12 weeks duration based on the parenteral administration of enrofloxacin and trimethoprim plus sulfadiazine, followed by the oral administration of doxycycline. Induction of immunosupression in six randomly chosen horses after completion of treatment did not lead to recrudescence of disease. CONCLUSION: This study demonstrates that long term treatment of glanderous horses with a combination of various antibiotics seems to eliminate the agent from the organism. However, more studies are needed to test the effectiveness of this treatment regime on B. mallei strains from different endemic regions. Due to its cost and duration, this treatment can only be an option in certain situations and should not replace the current “testing and culling” policy, in conjunction with adequate compensation to prevent spreading of disease

Prevalence and Hematology of Tick Borne Hemoparasitic Diseases in Equines in and Around Lahore

Abstract.-A total of 395 ticks infected equines (166 horses, 115 mules, 114 donkeys) were incorporated to study the prevalence and hematology of tick borne hemoparasitic diseases (TBHD) in equines from March 2012 to February 2013 in Lahore, Pakistan. In horses Theileriosis was the most prevalent (38/166; 22.89%) TBHD followed by Anaplasmosis (34/166; 20.48%), Babesiosis (32/166; 19.28%) and mixed infection (18/166; 10.84%). In mules, Babesiosis was the most prevalent (30/115; 26.09%) TBHD followed by mixed infection (25/115; 21.74%), Anaplasmosis (15/115; 13.04%) and Theileriosis (15/115; 13.04%). In donkeys, the most prevalent TBHD was Babesiosis (27/114; 23.68%) followed by Anaplasmosis (24/114; 21.05%), Theileriosis (21/114; 18.42%) and mixed infection (15/114; 13.16%). Statistical analysis revealed the significant difference (P&lt;0.05) among the species of TBHDs. All the equines showed that due to tick infestation there was a remarkable increase in TLC values and slightly increased in TEC values than the values from the healthy equines while PCV remained in the normal range in horses and mules with a significant association (p&lt;0.05) between them but values slightly increased in donkeys with significant difference in the values. There was an increase in Hb values in mules and donkeys but decrease in horses than the values from the healthy equines. According to the statistical analysis there was a significant difference (p&lt;0.05) in Hb and TLC values of all equines than the normal values of equines

Comparison of diagnostic tests for the detection of Brucella spp. in camel sera

<p>Abstract</p> <p>Background</p> <p>Brucellosis in livestock causes enormous losses for economies of developing countries and poses a severe health risk to consumers of dairy products. Little information is known especially on camel brucellosis and its impact on human health. For surveillance and control of the disease, sensitive and reliable detection methods are needed. Although serological tests are the mainstay of diagnosis in camel brucellosis, these tests have been directly transposed from cattle without adequate validation. To date, little information on application of real-time PCR for detection of <it>Brucella </it>in camel serum is available. Therefore, this study was performed to compare the diagnostic efficiency of different serological tests and real-time PCR in order to identify the most sensitive, rapid and simple combination of tests for detecting <it>Brucella </it>infection in camels.</p> <p>Findings</p> <p>A total of 895 serum samples collected from apparently healthy Sudanese camels was investigated. Sudan is a well documented endemic region for brucellosis with cases in humans, ruminants, and camels. Rose Bengal Test (RBT), Complement Fixation Test (CFT), Slow Agglutination Test (SAT), Competitive Enzyme Linked Immunosorbant Assay (cELISA) and Fluorescence Polarization Assay (FPA) as well as real-time PCR were used. Our findings revealed that <it>bcsp31 </it>kDa real-time PCR detected <it>Brucella </it>DNA in 84.8% (759/895) of the examined samples, of which 15.5% (118/759) were serologically negative. Our results show no relevant difference in sensitivity between the different serological tests. FPA detected the highest number of positive cases (79.3%) followed by CFT (71.4%), RBT (70.7%), SAT (70.6%) and cELISA (68.8%). A combination of real-time PCR with one of the used serological tests identified brucellosis in more than 99% of the infected animals. 59.7% of the examined samples were positive in all serological tests and real-time PCR. A subpopulation of 6.8% of animals was positive in all serological tests but negative in real-time PCR assays. The high percentage of positive cases in this study does not necessarily reflect the seroprevalence of the disease in the country but might be caused by the fact that the camels were imported from brucellosis infected herds of Sudan, accidentally. Seroprevalence of brucellosis in camels should be examined in confirmatory studies to evaluate the importance of brucellosis in this animal species.</p> <p>Conclusion</p> <p>We suggest combining <it>bcsp31 </it>real-time PCR with either FPA, CFT, RBT or SAT to screen camels for brucellosis.</p

Serologische Diagnostik von Rotz unter Berücksichtigung endemischer und nicht- endemischer Tierseuchensituationen

Glanders is a highly infectious and zoonotic disease of solipeds caused by Burkholderia mallei. Progressive loss of efficiency and fatal outcome resulted in massive economic losses which forced veterinary authorities throughout the world to implement disease control measures; these included mass testing using the complement fixation test and/or malleinisation, and the culling of positives. This led to the eradication of glanders from Western Europe and North America in the 1950s. However, in the last decade, the number of outbreaks in Asia and South America increased steadily and glanders regained the status of a re-emerging transboundary disease. Pakistan has been an endemic country for the past 120 years but concise data on the presence of disease are not available. A total of 533 serum samples were collected from draught equines, a suspected risk group for glanders, from various districts of the Pakistani Punjab. The complement fixation test (CFT) and the highly sensitive Western blot technique were used for serodiagnosis. No positive animal (horse, mule, and donkey) was found. Glanders seems to be restricted to remote, sporadic pockets of endemicity and may cause outbreaks after being introduced into naive populations by (asymptomatic) shedders. Various serological tests were used for the diagnosis of glanders in the past but still complement fixation test (CFT) is the internationally prescribed test for trading equines. A new immunoblot (IB) technique has recently been introduced to overcome the well known shortcomings of CFT i. e. a considerable number of false positive and negative results and anticomplementary effect of sera. The objective of this study was the comparative evaluation of two glanders CFT antigens commercially available at Central Veterinary Institute of Wageningen UR, Lelystad, The Netherland (CIDC) and at c.c.pro GmbH, Oberdorla, Germany (c.c.pro) in an glanders endemic area regarding specificity and sensitivity. A total of 1,678 serum samples from the endemic region (Province Punjab, Pakistan) and a non-endemic area (Germany) were analysed. All sera tested positive or suspicious with CFT were analysed by the confirmatory IB to exclude CFT false positive results. Both CFT antigens showed 100% sensitivity. The use of CIDC or c.c.pro antigen resulted in specificities of 77.45% or 75.71% for sera from endemic area and 93.75% or 94.79% for sera from non-endemic areas, respectively. The results demonstrate the different performances of identical tests in different epidemiologically settings. Based on these results, the combined use of CFT and IB is highly suggestive for the serodiagnosis of glanders. Good agreement was calculated between CFT (using either c.c.pro or CIDC antigen) and IB. In response to third objective of comparatively evaluation of three commercially available antigens: (c. c. pro, CIDC and USDA) using sera from glanders free (Germany) and glanderous/immunised animals, the sensitivity and specificity of three commercially available complement fixation test (CFT) antigens from c.c.pro (c.c.pro), Central Veterinary Institute of Wageningen UR (CIDC) and the United States Department of Agriculture (USDA) were comparatively evaluated by testing 410 sera collected from glanders-endemic and non-endemic areas (200 true negative randomly collected sera and 210 sera collected from experimentally immunised animals (12 rabbits, 19 horses), clinical-positive (135) and culture-positive (44) horses, donkeys and mules). Immunoblotting (IB) was used as gold standard test. Highest sensitivity was shown for the CIDC antigen (100%) followed by the c.c.pro antigen (99.39%). However, the USDA antigen showed substantially less (P<0.05) sensitivity (62.19%). Highest specificity was found for the USDA antigen (100%) followed by the CIDC (97.5%) and c.c.pro antigen (96.5%). Positive and negative predictive values for each antigen were calculated to be: 95.88 and 99.48 (c.c.pro), 97.04 and 100 (CIDC), 100 and 76.33% (USDA). Almost perfect agreement (0.96) was found between CFT using either c.c.pro or CIDC and IB. Due to almost perfect agreement (0.96), CFT using c.c.pro or CIDC antigen can be combined with IB to increase the detection rate of glanders among infected animals.Rotz ist eine anzeigepflichtige, hochkontagiöse Infektionskrankheit der Einhufer und wird durch das Gram-negative, unbewegliche Bakterium Burkholderia (B.) mallei verursacht. Der Ausbruch und die Anzeige der Erkrankung führen durch Handelssperren zu erheblichen ökonomischen Verlusten für die betroffenen Länder. Deshalb forcieren die Veterinärbehörden weltweit Überwachungsuntersuchungen mittels Komplementbindungsreaktion (KBR) und Malleintest. Durch eine strikt durchgeführte Ausmerzung positiver Tiere, konnte Rotz in Westeuropa und Nordamerika in den 50iger Jahren ausgerottet werden. Durch eine steigende Anzahl von Rotzerkrankungen in den letzten 10 Jahren in Südamerika und in Asien wird Rotz jedoch als „re-emerging“ Tierseuche eingestuft. Für die serologische Diagnostik von Rotz sind verschiedene Testverfahren beschrieben. Die KBR ist jedoch die für internationale Handelsuntersuchungen vorgeschriebene Methode. In der Diagnostik mit der KBR bereiten insbesondere falsch positive Ergebnisse oder Proben mit antikomplementären Eigenschaften immer wieder Probleme. Deshalb kam in der vorliegenden Arbeit zusätzlich zur KBR ein Immunoblot-Verfahren (IB) zur Anwendung. Pakistan gilt seit 120 Jahren als endemisch für Rotz. Präzise Daten über die gegenwärtige Situation sind jedoch nicht verfügbar. Deshalb wurden in einer vorläufigen Prävalenzstudie 533 Serumproben von Zugtieren (Pferde, Esel, Maultiere), einer potentiell verdächtigen Risikogruppe, in verschiedenen Bezirken der Provinz Punjab in Pakistan gesammelt und mit der KBR und einem hochsensitiven IB untersucht. Es wurden keine positiven Tiere nachgewiesen. Es kann geschlussfolgert werden, dass Rotz scheinbar nur in begrenzten, weiter abgelegenen Gebieten vorkommt und durch asymptomatische Ausscheider nur sporadisch in erregerfreie Populationen eingebracht wird. In einer weiteren Studie der vorliegenden Arbeit kamen ebenfalls die KBR und der IB zur Anwendung wobei insbesondere die Nutzung verschiedener diagnostischer KBR-Antigene unter Beachtung verschiedener endemischer Situationen bewertet wurde. Gegenstand dieser Studie war die vergleichende Evaluierung zweier Rotz- KBR-Antigene, kommerziell erhältlich bei Central Veterinary Institute in Wageningen UR, Lelystad, Niederlande (CIDC) und c.c.pro GmbH, Oberdorla, Deutschland (c.c.pro) hinsichtlich ihrer Spezifität und Sensitivität in einer Rotz-endemischen Region Pakistans. Insgesamt wurden 1678 Serumproben (davon 25 positive Proben von Tieren bei denen die Infektion durch die Isolierung des Erregers bestätigt wurde) aus der Provinz Punjab (Pakistan) und einer nicht endemischen Region (Deutschland) getestet. Alle KBR-positiven und -verdächtig reagierenden Proben wurden mittels IB untersucht, um falsch positive KBR- Ergebnisse auszuschließen. Beide KBR-Antigene zeigten eine Sensitivität von 100%. Die KBR unter Einsatz des CIDC- oder c.c.cpro-Antigens zeigte Spezifitäten von 77,45% und 93,75% oder 75,71% und 94,79% bei Anwendung auf Proben aus der endemischen oder nicht-endemischen Region. Die Ergebnisse zeigen, dass die Spezifität identischer serologischer diagnostischerverfahren, angewendet in Regionen mit unterschiedlicher epidemiologischer Lage erheblich schwanken kann. Für eine dritte Studie wurden drei kommerziell erhältliche KBR-Antigene zur Rotzdiagnostik, das c.c.pro- und CIDC-KBR-Antigen und das KBR-Antigen des United States Department of Agriculture (USDA), vergleichend eingesetzt. Das c.c.pro-Antigen und CIDC-Antigen bestehen aus einer Mischung dreier B. mallei Isolaten (Bogor, Zagreb und Mukteswar), wohingegen das USDA- Antigen aus nur einem B. mallei Isolat (Chinese) hergestellt wurde. Insgesamt wurden 410 Seren, gesammelt von 200 Pferden aus Deutschland (Gruppe I, negativ) und 210 Serumproben (Gruppe 2, positiv) getestet. Die Seren der Gruppe I galten als negativ, weil Rotz in Deutschland seit mehr als 50 Jahren ausgerottet ist. Die positive Gruppe II setzt sich zusammen aus 44 Tieren aus Pakistan, bei denen die Infektion durch die Isolierung des Erregers bestätigt wurde, aus 135 klinisch krank beurteilten Tieren aus Pakistan und Brasilien, 12 Seren stammten von einem immunisierten Kaninchen und 19 Seren von einem immunisierten Pferd. Die Durchführung der KBR erfolgte gemäß den Vorgaben des OIE - “Manual of Diagnostic Tests and Vaccines for Terrestrial Animals” unter Nutzung des kommerziell verfügbaren Komplement- und hämolytischen Systems der Firma Virion/Serion. Das c.c.pro- und CIDC-Antigen wurde mit dem Serum für 18h bei 4°C (Kältebindung) und das USDA-Antigen für 1h bei 37°C (Wärmebindung) inkubiert. Alle Proben wurden auch im IB analysiert. Die höchste Sensitivität erreichte das CIDC-Antigen (100%) gefolgt durch das c.c.pro-Antigen (99,39%). Das USDA-Antigen zeigte eine deutlich geringere (P<0,05) Sensitivität (62,19%). Die höchste Spezifität konnte mit dem USDA-Antigen (100%) erzielt werden, gefolgt vom CIDC-Antigen (97,50%) und c.c.pro-Antigen (96,50%). Dabei zeigten die KBR mit dem c.c.pro- oder CIDC-Antigen zusammen mit dem IB die höchste Übereinstimmung (0,96). Ausgehend von diesen Ergebnissen wird für die serologische Diagnostik von Rotz die Kombination aus KBR mit dem c.c.pro- oder CIDC-Antigen und dem IB empfohlen

Prevalence and Effect of Helminthiasis on Haematological Parameters in the Migratory Sparrows (Alauda arvensis) and Treatment with an Antihelmintic, Fenbendazole

Abstract.-The present study was conducted to investigate the prevalence of helminthic parasites of gastrointestinal tract (GIT) and its effect on blood picture in the captured migratory sparrows (Aluda arvensis). The parasitized sparrows were treated with a broad-spectrum anthelmintic i.e., panacur containing fenbendazole. On the basis of the cloacal swab samples seen under microscope, a prevalence rate of 56% (n=112) among all the two hundred migratory sparrows was observed. Half of these positive birds (n=56) in number were necropsied in order to recover the live parasites from the gastrointestinal tract. Live Heterakis gallinae and Ascaridia galli were recovered from 50% and 32% of the birds, respectively and 18% birds showed live mixed infection of both the parasites. None of the birds was parasitized with live trematode and/or cestode. The haematology revealed that total erythrocyte count, hemoglobin, lymphocytes and eosinophils were decreased and total leucocytic count was increased. From the remaining live, half of the parasitized birds i.e., 56 migratory sparrows, a treatment trial was conducted and an efficacy rate of 95% was observed 7 days post-medication

Sero-prevalence of Brucella abortus among dairy cattle and buffaloes in Pothohar Plateau, Pakistan

The sero-prevalence of brucellosis was investigated among different breeds of cattle and buffaloes in Islamabad Capital Territory (Id), Rawalpindi and Attock regions of Pakistan. A total of 2330 milk samples (1168 cattle and 1162 buffaloes) were screened for the presence of Brucella abortus antibodies using the milk ring test (MRT). Information related to animal type, urbanicity, sampling area and breeds were collected with the help of a pre-tested questionnaire on the day of sampling. The overall sero-prevalence was 6.9% in cattle and 6.6% in buffaloes. More seropositive animals were found in ICT compared to the other regions. The odds of brucellosis sero-positivity were higher among cross breed cattle and Nili-ravi buffaloes. This study is the first evidence of prevalence of Brucella abortus up to breed level in dairy cattle. and buffaloes in Pakistan