Evaluation of ELISA coupled with Western blot as a surveillance tool for Trichinella infection in wild boar (Sus scrofa)

Trichinella surveillance in wildlife relies on muscle digestion of large samples which are logistically difficult to store and transport in remote and tropical regions as well as labour-intensive to process. Serological methods such as enzyme-linked immunosorbent assays (ELISAs) offer rapid, cost-effective alternatives for surveillance but should be paired with additional tests because of the high false-positive rates encountered in wildlife. We investigated the utility of ELISAs coupled with Western blot (WB) in providing evidence of Trichinella exposure or infection in wild boar. Serum samples were collected from 673 wild boar from a high- and low-risk region for Trichinella introduction within mainland Australia, which is considered Trichinella-free. Sera were examined using both an ‘in-house’ and a commercially available indirect-ELISA that used excretory–secretory (E/S) antigens. Cut-off values for positive results were determined using sera from the low-risk population. All wild boar from the high-risk region (352) and 139/321 (43.3%) of the wild boar from the low-risk region were tested by artificial digestion. Testing by Western blot using E/S antigens, and a Trichinella-specific real-time PCR was also carried out on all ELISA-positive samples. The two ELISAs correctly classified all positive controls as well as one naturally infected wild boar from Gabba Island in the Torres Strait. In both the high- and low-risk populations, the ELISA results showed substantial agreement (k-value = 0.66) that increased to very good (k-value = 0.82) when WB-positive only samples were compared. The results of testing sera collected from the Australian mainland showed the Trichinella seroprevalence was 3.5% (95% C.I. 0.0–8.0) and 2.3% (95% C.I. 0.0–5.6) using the in-house and commercial ELISA coupled with WB respectively. These estimates were significantly higher (P < 0.05) than the artificial digestion estimate of 0.0% (95% C.I. 0.0–1.1). Real-time PCR testing of muscle from seropositive animals did not detect Trichinella DNA in any mainland animals, but did reveal the presence of a second larvae-positive wild boar on Gabba Island, supporting its utility as an alternative, highly sensitive method in muscle examination. The serology results suggest Australian wildlife may have been exposed to Trichinella parasites. However, because of the possibility of non-specific reactions with other parasitic infections, more work using well-defined cohorts of positive and negative samples is required. Even if the specificity of the ELISAs is proven to be low, their ability to correctly classify the small number of true positive sera in this study indicates utility in screening wild boar populations for reactive sera which can be followed up with additional testing.     

The effect of a pressure wrap (ThunderShirt®) on heart rate and behavior in canines diagnosed with anxiety disorder

The effect of moderate-to-deep pressure on soft tissue has been studied in both humans and nonhuman animals with positive results shown in reducing anxiety and promoting relaxation. Canine anxiety disorders are often treated with medications and behavior modification programming. Pressure wraps are sometimes used to treat canine anxiety, but the effects of this investigation have not been rigorously investigated. The aims of this study were to investigate the use of a pressure wrap (ThunderShirt®; ThunderWorks, Durham, NC) on heart rate and behavior in dogs diagnosed with an anxiety disorder. Subjects were 90 dogs (39 males and 51 females) that were diagnosed with Separation Anxiety or Generalized Anxiety Disorder. Dogs were randomly assigned to 1 of the 3 groups, namely Experimental Group 1 (dogs wore the ThunderShirt® per manufacturer's recommendations), Experimental Group 2 (dogs wore the ThunderShirt® loosely without pressure), and Control Group (dogs had no specific treatment). Average and maximum heart rates were measured at baseline and after 15 minutes alone in the kennel. Dogs were video-recorded and analysis completed for 12 behaviors, namely pacing, panting, yawning, tongue-flicking, drooling, elimination, barking, stress whining, howling, licking, door orientation, and calmness. The results were as follows: Dogs in Experimental Group 1 showed significantly less increase from baseline in average heart rate than the Control Group dogs (P < 0.001) and dogs in Experimental Group 2 (P < 0.001). Dogs in Experimental Group 1did not differ significantly from Control Group dogs in maximum heart rate when all dogs were considered, but did differ significantly from the Control Group (P = 0.04) when only those dogs not currently on anxiety medication were considered. There were no statistically significant differences among groups on behavioral outcomes, except that the dogs in the Control Group were significantly more likely to orient toward the door than the dogs in both Experimental Groups 1 (OR = 7.46, P = 0.01) and 2 (OR = 4.42, P = 0.04). Dogs in the Experimental Group 1 presented a trend of less tongue-flicking (P = 0.06) and yawning (P = 0.07) stress reduction behaviors when compared with the other 2 groups. More research should be conducted with the ThunderShirt® on behavioral outcomes. The ThunderShirt® can be used with behavior modification programming and medication as an adjunct treatment option to assist dogs diagnosed with anxiety disorder to reduce heart rate.     

Systemic and local immune response in pigs intradermally and intramuscularly injected with inactivated Mycoplasma hyopneumoniae vaccines

The systemic and respiratory local immune response induced by the intradermal administration of a commercial inactivated Mycoplasma hyopneumoniae whole-cell vaccine (Porcilis^® MHYO ID ONCE – MSD AH) in comparison with two commercial vaccines administered via the intramuscular route and a negative control (adjuvant only) was investigated. Forty conventional M. hyopneumoniae-free pigs were randomly assigned to four groups (ten animals each): Group A = intradermal administration of the test vaccine by using the needle-less IDAL^® vaccinator at a dose of 0.2 ml; Group B = intramuscular administration of a commercially available vaccine (vaccine B); Group C = intramuscular administration of the adjuvant only (2 ml of X-solve adjuvant); Group D = intramuscular administration of a commercially available vaccine (vaccine D). Pigs were vaccinated at 28 days of age. Blood and bronchoalveolar lavage (BAL) fluid samples were collected at vaccination (blood only), 4 and 8 weeks post-vaccination. Serum and BAL fluid were tested for the presence of antibodies by ELISA test. Peripheral blood monomorphonuclear cells (PBMC) were isolated to quantify the number of IFN-γ secreting cells by ELISpot. Moreover, cytokine gene expression from the BAL fluid was performed. Total antibodies against M. hyopneumoniae and specific IgG were detected in serum of intradermally and intramuscularly (vaccine B only) vaccinated pigs at 4 and 8 weeks post-vaccination. M. hyopneumoniae specific IgA were detected in BAL fluid from vaccinated animals (Groups A and B) but not from controls and animals vaccinated with the bacterin D (p < 0.05). Significantly higher gene expression of IL-10 was observed in the BAL fluid at week 8 post-vaccination in the intradermally vaccinated pigs (p < 0.05). The results support that the intradermal administration of an adjuvanted bacterin induces both systemic and mucosal immune responses. Moreover, the intramuscularly administered commercial vaccines each had a different ability to stimulate the immune response both systemically and locally.     

A prospective study on fitness,workload and reasons for premature training ends and temporary training breaks in two groups of riding horses

Little is known about wastage in riding horses and the factors like fitness and workload that may reduce injuries and maximise welfare. To evaluate fitness, workload and reasons for premature training ends (PTEs) and temporary training breaks (TTBs) during a nine week training period, two groups of riding horses were used: Group A consisting of 58 horses used for student equitation courses (32 with training prior to admission and 26 without) and Group B consisting of 26 horses owned by two riding schools (school-I and school-II). To assess fitness, all horses performed a standardised exercise test (SET) at the start (SET-I) and end of the training period (SET-II) measuring heart rate (HR bpm) and speed (m/s). In addition, all horses were monitored daily during the training period for their health and workload.In Group A, trained horses had significantly lower HRs in SET-I (P = 0.05) compared to untrained horses and in SET-II, trained horses tended to have lower HRs than untrained horses, though this was not statistically significant (P = 0.057). During the training period all horses received an identical workload. A total of 19.0% of Group A horses ended the training period prematurely for veterinary reasons (PTEV); of those untrained horses had earlier a PTEV in the training period (after 2.8 ± 1.3 weeks) than trained horses (after 4.1 ± 1.5 weeks, P = 0.030).In Group B, school-I and school-II horses did not differ significantly in fitness level nor in workload. More school-II horses ended the training period prematurely for veterinary reasons (n = 7; 70%) compared to school-I horses (n = 4; 25%, P = 0.032), although seven (63.6%) of these horses were still continuously used in riding lessons. In both groups (A and B), small injuries (without a temporary training break) were significantly associated with premature training ends for veterinary reasons later on: in Group A small injuries preceded 27.3% of the PTEVs (P = 0.005) and in Group B small injuries preceded 54.5% of the PTEVs (P = 0.030).In conclusion, as all horses in each subgroup had the same workload, the occurrence of PTEV seemed not associated with the workload. In Group A horses, level of fitness seems to be an important factor for the point in time injuries will occur during the training period. In all horses, injuries were more likely when a temporary training break was not taken following seemingly minor injuries. Since a lot of injured Group B horses were used in riding lessons against veterinary advice, this may indicate that riding school owners have different perception on welfare and if true this may cause serious welfare problems.     

Frequency of antibodies against Besnoitia besnoiti in Brazilian cattle

Besnoitia besnoiti is a cyst-forming parasite that has been associated with economic losses in Africa and Europe. Besnoitiosis is considered as a re-emergent disease in the European continent. It is unknown whether cattle are exposed to B. besnoiti in the Americas, thus the aim of this study was to serologically investigate antibodies against B. besnoiti in a total of 2014 cattle serum samples from two states from Brazil. All samples were evaluated by IFAT and part of the positive sera was tested by Western blot (WB) using tachyzoites extracts under non-reducing condition. A total of 3.48% (70/2014) of the tested sera reacted positively by IFAT with titers of 200 (85.7%), 400 (10%) and 800 (4.3%). When 47 positive samples were assessed by WB a range of antigens from 7 to 206 kDa was recognized by the IFAT-positive sera. The results are suggestive of exposure of Brazilian cattle to B. besnoiti due to the titers (≥200) observed for some sera using IFAT. However, the antigens recognized by the IFAT-positive animals did not completely match with the WB patterns previously described by other working groups. It is possible that Brazilian cattle are exposed to B. besnoiti strains with different antigenic composition of those described in the European and African continent. Further studies are needed to confirm the presence of B. besnoiti or other Besnoitia species in Brazilian cattle.     

NTPDase and 5′-nucleotidase as inflammatory markers in cattle naturally infected by Eurytrema coelomaticum

The aim of this study was to evaluate seric NTPDase and 5′nucleotidase activities of cattle naturally infected by Eurytrema coelomanticum, as well as to correlate them to histopathological lesions in the pancreas and the degree of parasitism. Blood samples and pancreas of 51 bovines were collected on a slaughterhouse in Southern Brazil: 33 from cattle naturally infected by E. coelomanticum (the Group A), and 18 from uninfected animals (the Group B). Infected animals showed an average of 532 parasites per pancreas. In the pancreatic histology, ducts displayed hyperplasia, stenosis, proliferation of fibrous tissue, and interstitial inflammatory infiltration of lymphocytes. The serum from infected animals showed an increase in NTPDase activity when ATP was used as substrate (P < 0.001). For the ADP substrate, there was no difference between groups regarding NTPDase activity (P = 0.37), as well as 5′-nucleotidase activity (P = 0.27). Correlating NTPDase activity (ATP substrate) with the degree of histopathological lesions (rho = 0.66, P < 0.001) and the parasitic load on the pancreas (rho = 0.65, P < 0.001), a positive correlation was observed. Similar results were found between the degree of histopathological lesions and NTPDase activity (ADP substrate; rho = 0.29, P = 0.03), and 5′nucleotidase activity (rho = 0.35, P = 0.01). Based on the results of NTPDase and 5′nucleotidase enzymes in cattle naturally infected by E. coleomanticum, it is possible to suggest that these enzymes are involved in the modulation of inflammation, and they can act as markers of inflammatory response.     

Mycoplasma ovis infection in goat farms from northeastern Brazil

Although Mycoplasma ovis (formerly Eperythrozoon ovis) has been described in small ruminants worldwide, data on M. ovis in goats remain scarce. Accordingly, the aims of the present study were to i) determine the prevalence of hemoplasmas in goats, ii) identify the tick species parasitizing the animals, and iii) determine factors associated with infection in five dairy and three beef goat farms from the Paraíba State, northeastern Brazil. Blood samples were obtained from 402 goats. Samples were screened for hemoplasmas using a pan-hemoplasma PCR. The positive samples were confirmed by sequencing. An epidemiological questionnaire was given to each farm owner addressing age, gender, and presence of ticks. A total of 158/402 (39.3%) goats were positive for M. ovis by PCR. Sequencing of PCR positive samples has shown ≥99% identity with multiple M. ovis 16S rDNA sequences deposited in GenBank, including M. ovis isolates from humans. Dairy (OR = 2.15; 95% CI: 1.40–3.32%; P = 0.0004) and anemic goats (OR = 2.33; 95% CI: 1.51–3.71%; P = 0.0001) were more likely to be infected than beef and non-anemic animals, respectively. Amblyomma parvum (49/52, 94.23%) and Rhipicephalus microplus (3/52, 5.77%) were the tick species found parasitizing the animals, with no significant association between the presence of ticks and infection by M. ovis (P = 0.1164). This is the first reportedly molecular detection of M. ovis infection in goats from South America. In conclusion, M. ovis is highly prevalent in goats from northeastern Brazil, mainly in dairy animals.     

Evaluation of peripheral lymphocytes after weaning and vaccination for Mycoplasma hyopneumoniae

This study evaluated immune cell populations in pigs following weaning and vaccination for Mycoplasma hyopneumoniae. Piglets (n = 24) were weaned (day 0) at 16 (±1) days of age, and randomly assigned to the vaccination group (n = 16) or control group (n = 8). Complete blood cell counts, flow cytometry and serology were completed for blood samples collected on days 0 (within hours of weaning), 3, 7, 14, 30 and 60. The M. hyopneumoniae S:P ratios (sample optical density: positive control optical density) were negative in the vaccination group until days 30 and 60, when the S:P ratios were 1.3 and 1.0, respectively. Control animals remained serologically negative. The percentage of CD4⁺ T cells was less (P < 0.01) in control pigs than vaccinated pigs at day 3. In contrast, numbers of CD8⁺ and CD4⁺CD8⁺ T cells were greater (P < 0.01) in control pigs than in vaccinated pigs at days 3 and 7. After day 7, few differences in immune cell types were evident between the groups. Differences in lymphocyte populations could not be solely attributed to vaccination, due at least in part, to the confounding influence of weaning. It was difficult to distinguish the influence of vaccination from the impact of weaning on peripheral immune cell populations.     

Apparent seroprevalence,isolation and identification of risk factors for brucellosis among dairy cattle in Goa,India

Brucellosis is a highly contagious zoonotic infection affecting livestock and human beings. The disease has been reported worldwide except in few countries where it has been eradicated. The prevalence of brucellosis among cattle from 11 farms having a history of abortions was studied. A total of 481 samples comprising of blood, milk, vaginal swabs, vaginal discharges, placental tissues and fetal tissues were collected from 296 animals. Clinical samples were processed for the isolation of Brucella. Serum samples (n = 296) were tested by Rose Bengal Plate Test (RBPT) and indirect ELISA. A total of 90 (30.40%) and 123 (41.55%) samples were positive by RBPT and indirect ELISA, respectively. Also 27.02% samples were positive by both the tests. Brucella isolates (n = 8) were recovered from clinical samples using Brucella selective media. All the isolates demonstrated PCR amplification for the bcsp31 and IS711 genes. Amplification of Brucella abortus specific primer was demonstrated by all the isolates in AMOS PCR indicating isolates to be of either B. abortus biotype 1, 2 or 4. Risk factors for transmission of brucellosis among cattle population were studied by field surveys. It was observed that lack of awareness about brucellosis (OR = 8.739, P = 0.138) and inadequate floor space (OR = 0.278, P = 0.128) were crucial risk factors for transmission of bovine brucellosis.     

Effect of supplemental sericea lespedeza leaf meal pellets on gastrointestinal nematode infection in grazing goats

Feeding sun-dried sericea lespedeza [SL; Lespedeza cuneata (Dum-Cours.) G. Don.] reduces gastrointestinal nematode (GIN) infection in goats fed in confinement, but effects of this forage when fed as a supplement to goats on pasture are unclear. A study was completed in which supplemental feeds (75 and 95% SL leaf meal pellets and a commercial pellet, all fed at 0.91 kg/head/day) were offered to thirty growing male Spanish goats (9 months old, 20.6 ± 2.8 kg, 10/treatment) grazing perennial warm-season grass pastures in Fort Valley, GA, from September to November, 2010. Fecal and blood samples were taken from individual animals weekly to determine fecal egg count (FEC) and packed cell volume (PCV), respectively, and animal weights were recorded at the start and end of the trial. After 11 weeks grazing, animals were slaughtered for recovery, counting, and speciation of adult GIN from the abomasum and small intestines. There was no difference in FEC between goats fed the 75 and 95% SL leaf meal pellets, but both groups had lower (P < 0.05) FEC than the goats fed the commercial pellets from days 35 to 77. The PCV values were not affected by the dietary treatments. Animal gain per day averaged 102.0, 77.2, and 53.3 g for goats fed 95% SL, commercial, and 75% SL pellets, respectively (P < 0.05). The 95% SL leaf meal pellet goats had 93.0 and 47.3% fewer (P < 0.05) total (male + female) adult Haemonchus contortus and Teladorsagia circumcincta, respectively, than control animals, while only male H. contortus were lower (47.6%; P < 0.05) in 75% SL-fed goats compared with commercial pellet-fed animals. Feeding supplemental SL leaf meal pellets improved animal performance (95% SL pellets) and reduced worm burdens (75 and 95% SL pellets) in young grazing goats and is a useful tool for natural GIN control in small ruminants.     

Clinical evaluation and antibody responses in sheep after primary and secondary experimental challenges with the mange mite Sarcoptes scabiei var. ovis

In this work the clinical evolution and the specific serum IgG and IgE antibody responses in sheep after primary (n = 10) and secondary (n = 4) experimental challenges with the mange mite Sarcoptes scabiei var. ovis were studied. The primary infection was characterized by the development of mange lesions in all sheep, a detection of live S. scabiei mites in 70% skin scrapings taken in week 10 post-challenge (PC), strongly raised and sustained specific IgG levels and a more moderate but continuous rise in specific IgE levels. Seroconversion was detected for IgG and IgE by ELISA in 90% and 60% of the sheep in week 8 PC, respectively. By Western-blotting (WB), ten IgG-reactive bands (36–120 kDa) and four IgE-reactive bands (90–180 kDa) were observed in week 8 PC. Following the secondary challenge the ewes developed a smaller area of mange lesion than that seen following primary challenge and live S. scabiei mites were not detected in skin scrapings collected in week 8 PC, suggesting that sheep had developed immunity to re-infection. Compared to primary infection, the specific IgG secondary antibody levels were transient, but in contrast there was an anamnestic IgE response, resulting in an elicitation of specific serum IgE levels in week 2 PC significantly higher than those demonstrated after primary infection. WB analysis revealed one additional IgG-reactive band (180 kDa) and no additional IgE-reactive bands. Determining the immunodiagnostic or vaccination value of the IgG-reactive antigens and IgE-reactive allergens detected requires further studies.     

Detection of trichinellosis in a historically Trichinella-free area of Argentina

The aim of the present work was to determine the presence of human and porcine trichinellosis in an area of Argentina historically regarded as Trichinella-free. Human blood donors (n = 216) and swine destined for consumption (n = 57) were evaluated by serological techniques (ELISA, immunofluorescence, and/or Western Blot). Muscle tissues from 26 of the pigs were evaluated for the presence of Trichinella larvae by the artificial digestion method. A questionnaire was used to collect and evaluate data on eating habits of the human population under study and on swine-raising conditions. The survey showed that 98.1% of the individuals (n = 212) were regular consumers of pork in the form of stuffed products such as sausages produced by local butchers. The seroprevalence (positive sera by at least two of the three methods) was 8.3% (n = 18) for human trichinellosis and 24.5% (n = 14) for porcine trichinellosis. Trichinella spiralis larvae were found in 2 of the 26 pigs (7.7%) with parasite loads of 0.33 and 2.4 muscle larvae per gram. Twelve swine found positive by serological and/or parasitological tests were raised under poor sanitary conditions (presence of rubbish in the surroundings, with cannibalism and scavenging behaviors, presence of rodents, etc.). Our study confirms the existence of porcine trichinellosis in an area regarded as Trichinella-free, provides supporting serological evidence of human infection in this area, and indicates that failure to report cases of trichinellosis based on inadequate surveillance can result in incorrect prevalence classification of an area.     

Neospora caninum DNA detection by TaqMan real-time PCR assay in experimentally infected pregnant heifers

Neosporosis has been considered the main cause of abortion between the first and the second trimester of pregnancy in cattle. Therefore, the objective of this study was to identify the presence of Neospora caninum DNA obtained from experimental models based on the evaluation of different areas of the fetal nervous system and organs from heifers previously inoculated with NC-1 after or before insemination. This study was performed with Hereford × Nelore (n = 29) heifers and all animals were considered free of diseases at the beginning of the experiment. All animals were bred by fixed-time artificial insemination (TAI) and allocated as follows: (a) seronegative heifers subjected to TAI (TAI, n = 9), (b) heifers infected with N. caninun 60 days prior to TAI (NC-1 + TAI, n = 9), and (c) heifers submitted to TAI and infected with N. caninum 60 days later (TAI + NC-1, n = 11). The pregnancy was confirmed by transrectal ultrasonography 35 days after TAI and evaluated every 30 days until the end of gestation. Fetuses were collected surgically at 170 days of gestation, and immediately necropsied to remove tissues aseptically. Samples of the central nervous system (CNS), heart, kidney, lung, liver, skeletal muscle and caruncle were collected for DNA extraction. Days of gestation at abortion and interval from abortion to first insemination were examined by Student's t-test. At 35 days of gestation the pregnancy rates in the group NC-1 + TAI (4/9, 44.4%) was lower than in the control group (8/9, 88.8%, P < 0.05). At 60 days, the pregnancy rates in the NC-1 + TAI group (0/4, 0%) was lower compared to TAI + NC-1 (5/7, 71.4%) and control (6/8, 75.0%) groups (P < 0.05). Animals from the group NC-1 + TAI were re-inseminated 60 days after the first TAI. After pregnancy losses throughout the study, 5 animals (TAI), 3 animals (NC-1 + TAI) and 5 animals (TAI + NC-1) maintained pregnancy until 170 days of gestation. TaqMan RT-PCR demonstrated the presence of N. caninum DNA in the medulla and right posterior cortex in 3 out of 5 fetuses from the TAI + NC-1 group. We concluded that heifers infected after TAI had a higher incidence of the parasite at the fetus CNS. Identification of N. caninum by TaqMan RT-PCR would assist in the investigation of infection and in the evaluation of vaccines or therapeutic drugs to control neosporosis in cattle.     

Vaccination with a genotype 1 modified live vaccine against porcine reproductive and respiratory syndrome virus significantly reduces viremia,viral shedding and transmission of the virus in a quasi-natural experimental model

The present study assessed the efficacy of vaccination against genotype 1 porcine reproductive and respiratory syndrome virus (PRRSV) in terms of reduction of the transmission. Ninety-eight 3-week-old piglets were divided in two groups: V (n = 40) and NV (n = 58) that were housed separately. V animals were vaccinated with a commercial genotype 1 PRRSV vaccine while NV were kept as controls. On day 35 post-vaccination, 14 NV pigs were separated and inoculated intranasally with 2 ml of a heterologous genotype 1 PRRSV isolate (“seeder” pigs, SP). The other V and NV animals were distributed in groups of 5 pigs each. Two days later, one SP was introduced into each pen to expose V and NV to PRRSV. Sentinel pigs were allocated in adjacent pens. Follow-up was of 21 days. All NV (30/30) became viremic after contact with SP while only 53% of V pigs were detected so (21/40, p < 0.05). Vaccination shortened viremia (12.2 ± 4 versus 3.7 ± 3.4 days in NV and V pigs, respectively, p < 0.01). The 50% survival time for becoming infected (Kaplan–Meier) for V was 21 days (CI_95% = 14.1–27.9) compared to 7 days (CI_95% = 5.2–8.7) for NV animals (p < 0.01). These differences were reflected in the R value as well: 2.78 (CI_95% = 2.13–3.43) for NV and 0.53 (CI_95% = 0.19–0.76) for V pigs (p < 0.05). All sentinel pigs (10/10) in pens adjacent to NV + SP pens got infected compared to 1/4 sentinel pigs allocated contiguous to a V + SP pen. These data show that vaccination of piglets significantly decrease parameters related to PRRSV transmission.     

The prevalence of Giardia infection in dogs and cats,a systematic review and meta-analysis of prevalence studies from stool samples

Giardia has a wide range of host species and is a common cause of diarrhoeal disease in humans and animals. Companion animals are able to transmit a range of zoonotic diseases to their owners including giardiasis, but the size of this risk is not well known. The aim of this study was to analyse giardiasis prevalence rates in dogs and cats worldwide using a systematic search approach. Meta-analysis enabled to describe associations between Giardia prevalence and various confounding factors. Pooled prevalence rates were 15.2% (95% CI 13.8–16.7%) for dogs and 12% (95% CI 9.2–15.3%) for cats. However, there was very high heterogeneity between studies. Meta-regression showed that the diagnostic method used had a major impact on reported prevalence with studies using ELISA, IFA and PCR reporting prevalence rates between 2.6 and 3.7 times greater than studies using microscopy. Conditional negative binomial regression found that symptomatic animals had higher prevalence rates ratios (PRR) than asymptomatic animals 1.61 (95% CI 1.33–1.94) in dogs and 1.94 (95% CI 1.47–2.56) in cats. Giardia was much more prevalent in young animals. For cats >6 months, PRR = 0.47 (0.42–0.53) and in dogs of the same age group PRR = 0.36 (0.32–0.41). Additionally, dogs kept as pets were less likely to be positive (PRR = 0.56 (0.41–0.77)) but any difference in cats was not significant. Faecal excretion of Giardia is common in dogs and slightly less so in cats. However, the exact rates depend on the diagnostic method used, the age and origin of the animal. What risk such endemic colonisation poses to human health is still unclear as it will depend not only on prevalence rates but also on what assemblages are excreted and how people interact with their pets.     

Entamoeba infections in different populations of dogs in an endemic area of Lahore,Pakistan

Entamoeba histolytica, a protozoan parasite that affects humans and other primates all over the world. It is a common waterborne pathogen in endemic areas that have fecal oral transmission cycle. The aim of the present study was to examine the prevalence of E. histolytica and other Entamoeba species cysts in three different dog populations. Fecal samples from 600 dogs were collected and processed to detect Entamoeba cysts using the triple fecal test (light microscopy) and fecal antigens of E. histolytica were detected using a fecal antigen ELISA (TechLab E. histolytica II). Because it is impossible to differentiate E. histolytica from Entamoeba dispar and E. moshkovskii, using light microscopy we referred to all cysts morphologically consistent with E. histolytica as E. histolytica/dispar/moskovskii to reflect this uncertainty. Samples from 197 household dogs without clinical signs, 122 samples from household dogs exhibiting clinical signs of diarrhea, dysentery and vomiting and 281 stray dogs with no specific clinical signs were examined. Entamoeba histolytica-like cysts were observed in 94 (15.6%, 95% CI = ±3.88) by triple fecal test microscopy and E. histolytica antigens were demonstrated in 66 (11%, 95% CI = ±4.41) by fecal antigen ELISA in 600 fecal samples. Significant differences (P ≤ 0.05) in prevalence were found between the three populations. Twenty (10.1%, 95% CI = ±7.86) and 11 (5.6%, 95% CI = ±7.70) of 197 fecal samples from household dogs without clinical signs were positive by microscopy and by antigen ELISA, respectively. Twenty-nine (23.8%, 95% CI = ±6.58) and 23 (18.8%, 95% CI = ±7.81) of 122 the fecal samples from household dogs with clinical signs were positive by microscopy and by antigen ELISA, respectively. Forty-five (16.01%, 95% CI = ±5.62) and 32 (11.3%, 95% CI = ±6.38) of 281 fecal samples from stray dogs were positive by microscopy and by fecal antigen ELISA, respectively. Dogs from the youngest age group (6 months to 1 year) were more likely to be E. histolytica antigen positive than were dogs from the other two older age groups, with a significant difference (P ≤ 0.05) between all age groups. Statistically, no significant (P ≥ 0.05) difference of prevalence was seen in male and female dogs. The local dogs had the highest prevalence rate of E. histolytica antigens (36 of 246, 14.2%, 95% CI = ±6.32) followed by imported breeds (11 of 115, 9.5%, 95% CI = ±10.4) and crossbred (19 of 239, 8.3%, 95% CI = ±7.47), indicating a significant (P ≤ 0.05) trend of positivity between various breeds of dogs. These findings suggest that dogs may play an important role in the epidemiology of this pathogen.     

Gasterophilus spp. infections in horses from northern and central Kazakhstan

A cross-sectional survey was performed to obtain current data on the gastrointestinal myiasis of horses in the provinces of Kostanay, Akmola and Karagandy, northern and central Kazakhstan. The stomach, small intestine and rectum of 148 slaughter horses were examined for Gasterophilus spp. larvae during a 26-month study period. All horses were infected with 2nd and 3rd stage larvae (mean intensity: 803 ± 350), and 22% of them harboured >1000 Gasterophilus spp. larvae each. Four species were identified: G. intestinalis (prevalence: 100%; mean intensity: 361 ± 240 larvae), G. haemorrhoidalis (100%; 353 ± 191), G. nasalis (100%; 73 ± 36) and G. pecorum (91.2%; 18 ± 10). Horses aged < 2 years were higher infected with Gasterophilus larvae than 2–4 years old animals. Both the prevalence and extremely high intensity of Gasterophilus infections of horses in these Kazakh regions suggest respective control measurements to improve the health and performance of the animals and to increase the economic income of horse owners.     

Evaluation of long-term antibody responses to two inactivated bovine viral diarrhoea virus (BVDV) vaccines

The aim of the present study was to determine the serological response of heifers after vaccination with two inactivated bovine viral diarrhoea virus (BVDV) vaccines by means of various ELISA tests. Three dairy farms were selected from the Galicia region of Spain. In each herd, a batch of heifers to be vaccinated for the first time was selected and followed for 15 months. Heifers from farm 1 (n = 25) were vaccinated with Vaccine A, whereas heifers from farm 2 (n = 16) were vaccinated with Vaccine B. Heifers from farm 3 (n = 17), where no BVDV vaccines were used, acted as controls. Blood samples were analyzed periodically for BVDV antibodies, using five commercial ELISAs, based on BVDV p80 antigen or whole virus.At the end of the study, none of the animals vaccinated with Vaccine A seroconverted according to p80 antibody status, whereas up to 80% tested positive by ELISA against whole virus antigen. For the animals vaccinated with Vaccine B, 2/16 animals seroconverted according to p80 antibody ELISAs, whereas all had seroconverted according to the ELISA against whole virus antigen. In most cases, based on the use of ELISAs to detect specific antibodies against the p80 protein, at 15 months post-vaccination with inactivated BVDV vaccines the responses did not seem to interfere with detection of antibody to BVDV infection. However, the finding of a small proportion of vaccinated animals seropositive against BVDV p80 antigen suggests that antibodies that interfere with diagnosis of BVDV infection within the herd could exist, even when using p80 ELISAs.     

Risk factors for Salmonella spp in Portuguese breeding pigs using a multilevel analysis

Salmonella is the second most frequent cause of foodborne illness in the European Union (EU), so EU enforced legislation to achieve a reduction in Salmonella prevalence in the swine sector. To set the reduction target each country carried out a baseline survey to estimate Salmonella prevalence. The aim of our study was to identify risk factors for the presence of Salmonella in breeding pigs based on the data of the Baseline Study for Salmonella in Breeding Pigs in Portugal. In total, 1670 pen fecal samples from 167 herds were tested by culture and 170 samples tested positive. Along with the collection of the samples a survey was applied to collect information about the herd management and potential risk factors. Multilevel analysis was applied to the data using generalized linear mixed models and a logit link function. The outcome variable was the presence/absence of Salmonella in the pen fecal samples. The first level was assigned to the pen fecal samples and the second level to the herds. The results showed significant associations between Salmonella occurrence and the factors (p < 0.05): maternity pens versus mating pens (OR = 0.39, 95%CI: 0.24–0.63), feed from external or mixed source versus home source (OR = 2.81, 95%CI: 1.19–6.61), more than 10 animals per pen versus 10 animals per pen (OR = 2.02, 95%CI: 1.19–3.43), North Region versus Alentejo Region (OR = 3.86, 95%CI: 1.08–13.75), rodents control (OR = 0.23, 95%CI: 0.090–0.59), more than 90% of boars homebred or no boars versus more than 90% of boars from an external source (OR = 0.54, 95%CI: 0.3–0.97), semen from another herd versus semen from insemination centers (OR = 4.47, 95%CI: 1.38–14.43) and herds with a size of 170 or more sows (OR = 1.82, 95%CI: 1.04–3.19). This study offers very relevant information for both the Portuguese veterinary authorities and the pig farmers currently developing control programmes for Salmonella. This is the first study providing evidence for semen and boars source as risk factors for Salmonella in breeding pigs.     

An unmatched case controlled study of clinicopathologic abnormalities in dogs with Bartonella infection

We compared clinicopathologic findings in dogs with Bartonella infection to Bartonella spp. negative dogs suspected of a vector-borne disease. Cases (n = 47) and controls (n = 93) were selected on the basis of positive or negative enrichment culture PCR results, respectively. Signalment, clinicopathologic findings and treatments were extracted from medical records. DNA sequencing identified Bartonella henselae (n = 28, 59.6%), Bartonella vinsonii subsp. berkhoffii (n = 20, 42.6%), Bartonella koehlerae (n = 3, 6.4%), Bartonella volans-like (n = 3, 6.4%) and Bartonella bovis (n = 1, 2.1%). There were no significant differences in age, breed, size, sex or neuter status between cases and controls. Dogs infected with Bartonella sp. often had a history of weight loss [OR = 2.82; 95% CI: 1.08–7.56] and were hypoglobulinemic [OR = 4.26; 95% CI: 1.31–14.41]. With the exception of weight loss and hypoglobulinemia, clinicopathologic abnormalities in Bartonella-infected dogs in this study were similar to dogs suspected of other vector-borne infections.