Methods for investigating Trichinella infections in domestic and wild animals

Trichinellosis is an important parasitic zoonosis that is caused by the intracellular nematode Trichinella spp.. Infection of humans occurs through consumption of raw (or undercooked) meat containing infectious larvae. In Europe, meat from pork, horse, and wild boar have been identified as most important sources of Trichinella infections in humans. In Switzerland, both the domestic pig and wild boar population are considered free of Trichinella. Conversely, Swiss foxes, lynxs and recently a wolf were found to be infected, the species identified in these animals was always referred to as Trichinella britovi. Although this species rarely infects pork and, compared to Trichinella spiralis, only causes reduced pathogenic effects in humans, the basic presence of Trichinella in Switzerland cannot be neglegted. This fact has gained increasing importance since the responsible authorities in the European Union (EU) are preparing regulations for the official Trichinella-control in meat in order to improve food safety for consumers. These regulations will be implemented as a consequence of the recent association of east European countries with the EU. This new legislation particularly takes into account, that in the past by far most cases of human trichinellosis in the EU were due to consumption of imported east European meat.Within the framework of the bilateral agreements of Switzerland with the EU, the Swiss veterinary public health authorities will have to comply with the foreseen EU regulations. Although diagnostic methods for the direct demonstation of Trichinella in pork meat are already routine practice in several Swiss abattoirs, the implementation of a meat control program for Trichinella for the entire slaughter pig population of the country would lead to an enormous increase in costs for the administration and will require an increased infrastructure in veterinary services. In order to find a reduced testing format for monitoring Trichinella infections in Swiss pork, an infection risk-oriented survey strategy is currently evaluated. In the present article, this minimized survey strategy is discussed regarding its compatibility with the EU regulations laying down rules for the official control of meat for Trichinella.     

Survey of Cryptosporidium spp. and Giardia spp. infections in various animals at a zoo in Japan.

A total of 284 fecal samples of 89 species (43 mammalian species and 46 avian species) were examined for Cryptosporidium oocysts and Giardia cysts from 1999 to 2002. Each sample was collected at the zoo located at Osaka in Japan and examined by microscopy after performing the sucrose flotation method and by two immunofluorescent assay kits for detection of Cryptosporidium oocysts and Giardia cysts. Cryptosporidium spp. was found only in a raccoon dog (Nyctereutes procyonoides), and Giardia spp. was detected in a mandarin duck (Aix galericulata) and two ruddy shelducks (Tadorna ferruginea). In this study, the prevalences of these parasites were found to be low. However, these results suggested that the infected animals could serve as a source of contamination for surface water. This is the first report about the survey of Cryptosporidium spp. and Giardia spp. at a zoo in Japan.     

Epidemiological investigations on Trichinella infections in farmed fur animals of Estonia

Farming of fur animals represents an important income in Estonia. Even though Trichinella worms does not induce a symptomatic disease in carnivores, the carcasses of skinned animals can increase the biomass of the parasite in the environment, if they are not properly destroyed. The aim of the present survey was to study the prevalence of Trichinella worms in farmed fur animals of Estonia. Of 281 muscle samples from blue foxes (Alopex lagopus), silver foxes (Vulpes vulpes fulva), minks (Mustela vison) and raccoon dogs (Nyctereutes procyonoides), which were collected in eight farms, Trichinella larvae have been detected in 21 animals (8%) from two farms by HCl-pepsin digestion. The highest number of larvae per gram of muscle was found in the front limb muscles. Larvae of the 21 isolates have been identified as Trichinella britovi or Trichinella nativa by a multiplex-PCR analysis.     

Experimental Trichinella infections in ponies.

Light Trichinella infections were established in three ponies given 1000, 5000 and 25000 T. spiralis spiralis infective larvae respectively by stomach tube. A predilection site of infection in all ponies was the tongue. Anti-Trichinella antibodies were detected in all ponies by the enzyme-linked immunosorbent assay using a T. spiralis spiralis excretory-secretory antigen. The ponies given 5000 and 25000 infective larvae reacted positively on days 26 and 24 postinfection, respectively, using a criterion of greater than or equal to 5 X mean optical density readings of preinfection sera as positive. The pony given 1000 larvae did not react positively although antibodies were present as indicated by 3 X to 4 X mean optical density readings of preinfection sera. The results of this limited study indicate that the enzyme-linked immunosorbent assay cannot be used to certify horsemeat free of Trichinella since the presence of detectable antibody levels appears to be related to the magnitude of the infection and duration of the infection when the animal is tested.     

Muscle distribution of sylvatic and domestic Trichinella larvae in production animals and wildlife

Only a few studies have compared the muscle distribution of the different Trichinella genotypes. In this study, data were obtained from a series of experimental infections in pigs, wild boars, foxes and horses, with the aim of evaluating the predilection sites of nine well-defined genotypes of Trichinella. Necropsy was performed at 5, 10, 20 and 40 weeks post inoculation. From all host species, corresponding muscles/muscle groups were examined by artificial digestion. In foxes where all Trichinella species established in high numbers, the encapsulating species were found primarily in the tongue, extremities and diaphragm, whereas the non-encapsulating species were found primarily in the diaphragm. In pigs and wild boars, only Trichinella spiralis, Trichinella pseudospiralis and Trichinella nelsoni showed extended persistency of muscle larvae (ML), but for all genotypes the tongue and the diaphragm were found to be predilection sites. This tendency was most obvious in light infections. In the horses, T. spiralis, Trichinella britovi, and T. pseudospiralis all established at high levels with predilection sites in the tongue, the masseter and the diaphragm. For all host species, high ML burdens appeared to be more evenly distributed with less obvious predilection than in light infections; predilection site muscles harbored a relatively higher percent of the larval burden in light infections than in heavy infections. This probably reflects increasing occupation of available muscle fibers as larger numbers of worms accumulate. Predilection sites appear to be influenced primarily by host species and secondarily by the age and level of infection.     

Intestinal establishment and reproduction of adult Trichinella spp. in single and mixed species infections in foxes (Vulpes vulpes)

Intestinal establishment and reproduction of adult Trichinella spiralis, Trichinella nativa, Trichinella britovi and Trichinella pseudospiralis were examined as single species or mixed species infections in foxes. This is the first study of intestinal dynamics of Trichinella spp. in a carnivore model and the results suggest that the intestinal phase is relatively short as only very few worms were recovered 10 days post-inoculation (dpi). In mixed species infection with equal doses of T. nativa and T. spiralis, molecular typing demonstrated that 64% of the intestinal worms and 78% of the muscle larvae were T. nativa. Conversely, T. spiralis dominated in the mixed species infections with T. pseudospiralis, constituting 66% of the intestinal worms and 94% of the muscle larvae. Although, the individual recoveries of intestinal worms were only up to 5.6% on day 1, and up to 1.5% on day 4 post-infection, the muscle larvae establishment was comparable to other fox studies. Infectivity, measured as muscle larvae burden did not differ among the four species of Trichinella, which is in contrast to other models with mice, rats, pigs or herbivores. Although statistically significant differences in intestinal worm burdens were found for some days, no distinct species were recovered in consistently higher numbers than the others.     

Prevalence of Trichinella spp. in North Spain wild fauna and new variety of Trichinella britovi identification

Trichinella spiralis and Trichinella britovi live in apparent sympatry among wild fauna of the Iberian Peninsula. In the present study 105 Trichinella isolates from wild mammals were typed by inter-sequence simple repeat PCR (ISSR-PCR). All isolates identified as T. spiralis were indistinguishable from the ISS48 reference strain. Among those belonging to T. britovi, four variations were clearly distinguishable; two of them, ISS11 C-76 and ISS86 MON, had been previously detected while the ISS2 reference strain and Trichinella Rioja 3, (MVUL/SP/02/R3) had not been reported before. The newly distinguished genotype of T. britovi was analyzed by ISSR-PCR, multiplex-PCR, UARR sequencing, and single larva cross-breeding with the other T. britovi genotypes including Trichinella T8 (ISS49). Among all of them, the ISS11 and ISS2 isolates were found to be the most frequent. The uniformity found within T. spiralis isolates is consistent with its recent introduction in Iberian Peninsula, whereas the presence of four variations within T. britovi suggests that this species is an endemic species. Orographical diversity of the West-End of Eurasian Region could act to preserve population diversity observed within T. britovi.     

Pathogenesis and serodiagnosis of experimental Trichinella spiralis spiralis and Trichinella spiralis nativa infections in cattle.

Trichinella spiralis spiralis infections were established in cattle by gavage and by feeding infected musculature in the ration. Trichinae were present in greatest numbers in masseter, tongue and diaphragm. Trichinella spiralis nativa had a low infectivity to cattle although a light infection was established in one cow by a heavy challenge. Cattle had an aversion to eating musculature unless it was camouflaged with molasses. Clinical signs of reluctance to eat and masticate were observed between 10 and 30 days postinfection. Eosinophil counts started to increase at seven days and peaked at about 30 days postinfection. By day 60 eosinophil counts returned to near preinfection levels but in animals examined greater than 90 days postinfection, the counts were variable. Focal lesions of eosinophilic myositis were observed up to about 90 days postinfection. Little cellular reaction was observed surrounding trichinae after muscle invasion and cyst development was completed except for cysts undergoing disintegration. Seroconversion occurred in all cattle examined between 7 and 14 days postinfection. Seroconversion was associated with IgG1 and IgG2 immunoglobulins. Peak levels of antibody occurred between 30 and 60 days. Cattle examined at 182 and 369 days postinfection showed a gradual decrease in antibody levels over time.     

Detection of Trichinella infection in food animals

The first part of this review article deals with classical methods used for the detection of Trichinella larvae in muscle samples of those animal species which are recognized as traditional sources of trichinellosis for human beings, as well as those species which are important for epidemiological reasons. Special consideration is given to the main applications of these methods (routine slaughter inspection, and epidemiological studies in reservoir animals), and to the major factors that may influence detection methods (sampling site, sample size). Historical, current and future aspects concerning national and EU legislation for Trichinella inspection are also presented. The latter part of this review is directed at serodiagnostic methods for the detection of Trichinella-specific antibodies in different animal species. Classical methods of serodiagnosis such as the complement fixation test and immunofluorescence antibody test are reviewed and the characteristics and performance of the ELISA are discussed. Factors dependent upon the animal species being tested or on components of the ELISA test system are considered. This paper also reviews systematic development of the ELISA in relation to improvements in test specificity and sensitivity. Additionally, remarks are made on implementing this test for surveillance and control programs in domestic pigs and wildlife.     

Retroviral infections of small animals.

Retroviral infections are particularly important in cats, which are commonly infected with feline leukemia virus and feline immunodeficiency virus. This article describes the biology of these viruses and explores current issues regarding vaccination and diagnosis. The seeming lack of a recognized retrovirus infection in dogs is speculated on, and current and potential future therapies are discussed.     

Prevalence of Sarcocystis spp. in meat-producing animals in Iraq.

The prevalence of Sarcocystis spp. infection was investigated in 605 sheep, 826 goats, 1080 cattle, 580 water buffaloes and 36 camels slaughtered from 1992 to 1996 in the Baghdad area (Iraq) using naked eye examination for macroscopic sarcocysts, and peptic digestion, muscle squash, squeezing methods and indirect fluorescent antibody test (IFAT) for microscopic types. The intestinal stages of the parasite were also studied in dogs experimentally fed with tissues containing microscopic cysts. The percentage prevalence of macroscopic cysts were 4.1, 33.6, 0.2, 15.6 and 0, and of the microscopic type, 97.0, 97.4, 97.8, 82.9 and 91.6 for the above-mentioned hosts, respectively. Among the different organs examined, macroscopic cysts were found to be highest in the oesophagus and the lowest in the heart. Peptic digestion method gave the highest rate (93.3%) followed by indirect fluorescent antibody test (IFAT) (88.6%), squeezing (81.3%), and muscle squash (81.2%). Each infected dog shed a total of about 150-200 million sporocysts. Histologically, developmental stages of the parasite were detected in the small intestinal mucosa of the dogs on Days 7 and 13 post-infection.     

Inhibited development of Ostertagia spp. and Cooperia spp. in naturally acquired infections in calves

Abstract

Aujeszky? disease is predominantly a nervous disease of piglets, weaners, and fatteners in which the mortality may vary from 540%. Respiratory disease is also a feature of infection with certain strains of the virus. Infection of pregnant sows may cause abortion, stillbirths, and mummification, and infertility may result in both boars and sows. Traditionally the serum neutralisation test has been used as an indicator of past infection with Aujeszky s's disease virus (ADV). However, cell-mediated immunity has recently been shown to play an important role in recovery and protection from infection with ADV and antibody-dependent cell-mediated cytotoxicity can be used as an alternative diagnostic test for detection of antibody. A number of inactivated, and live, avirulent vaccines are now available for control of the disease but their use is contraindicated in countries such as New Zealand which have a low prevalence of infection. Where possible early eradication is the best policy to follow.     

Towards a risk-based surveillance for Trichinella spp. in Danish pig production

Increasing demands for cost-effectiveness in surveillance for human health hazards from animal origins can be met by introducing risk-based principles. This e.g. implies targeting subpopulations with higher risk of infection compared to the whole population. Furthermore, historical data from surveillance can be collated and used to assess future risk of infection. To demonstrate the effectiveness of combining these two approaches, we used a model called “Discounting historical evidence”. It depends mainly on the annual risk of introduction (PIntro) and the surveillance system sensitivity (SSe) (ability to detect infection if present). The model implies simulations that reiterate for a number of years. For each year the output is updated with the confidence on absence in infection. Trichinella spiralis infection in pigs is used as an example. In Denmark, more than 20 million pigs are tested annually. Despite more than 70 years of testing no pigs have been found positive for Trichinella. Hence, PIntro is low. SSe can be estimated from the maximum number of infected carcasses expected under the specified design prevalence, and the sensitivity of the test applied. According to our assessment, the current prevalence of Trichinella in Danish pigs is less than one case per million, which we interpret as negligible risk. Based on this, a risk-based surveillance programme for Trichinella is designed that targets all out-door reared pigs as well as all sows and boars (current total 610,000 slaughtered annually). These subpopulations are judged to have higher risk of getting Trichinella. Again, SSe and PIntro are estimated and the model results show that risk-based surveillance can be applied without jeopardizing human health. Finally, we incorporate wildlife surveys and test quality assurance in the programme. The results of the simulation model were included in an application to the European Commission concerning Denmark's status as a region with negligible risk of Trichinella. In July 2007, the European Commission granted status as “negligible risk” to Danish pigs and pork.     

Trichinella species circulating among wild and domestic animals in Romania

Trichinellosis is one of the most important zoonotic diseases in Romania. Even though the disease is a serious public health concern, only a limited number of Trichinella isolates have been identified at the species level; in the past, all larvae were assumed to be Trichinella spiralis. The present study was conducted to identify Trichinella spp. circulating among wild and domestic animals in Romania, using PCR-based methods. Trichinella spp. larvae originating from 54 wild and 23 domestic mammals were examined. No Trichinella spp. larvae were detected in muscle samples of 182 birds. T. spiralis and Trichinella britovi were the only two species identified in the 40 isolates that yielded a positive PCR result. Overall, T. britovi was more prevalent (n = 26; 65%) than T. spiralis (n = 14; 35%). T. spiralis was the predominant species found in domestic animals (n = 9; 75%), while T. britovi was more prevalent in wildlife (n = 24; 86%). No mixed infections were found. The highest prevalence of Trichinella infection was detected in wolves (11/35; 31%), in European wild cats (4/28; 14%), and in red foxes (5/71; 7%). The distribution of Trichinella spp. in Romania does not show a species-specific clustering; both of the two species found were present over the entire range of counties studied.     

Infection with Cryptosporidium spp. in humans and cattle in Manitoba.

Between October 1, 1983 and October 31, 1984, fecal specimens from 3656 persons with enteritis and 182 calves, representing 148 herds having a neonatal diarrhea problem, were examined for oocysts of Cryptosporidium spp. Oocysts were found in 1% of human and 25% of bovine specimens. All infected persons were immunocompetent. Children under five years of age had an infection rate of 25/100,000 compared to 1.4/100,000 in older people (p less than 0.005). Rates in northern communities were four to seven times as high as those in southern Manitoba. Human infections occurred most commonly in late summer and fall. In beef calves infection occurred in winter and spring, the calving season in Manitoba. Epidemiological association between the infection in people and in cattle could not be established.