Limit of detection of Toxocara canis larvae in experimentally contaminated bovine milk

Abstract

This study aimed to evaluate the limit of detection of Toxocara canis larvae in experimentally contaminated commercial bovine milk samples, based on a centrifuge-sedimentation technique. Firstly, bovine milk (whole and skim) samples were contaminated with 50 T. canis larvae in order to evaluate the interference of milk fat with the recovery of the larvae. Next, the effects of 10% formalin (100 µL), ether (100 µL), and a combination of both solutions on the recovery of the larvae was examined. Thereafter, the limit of detection of the larvae was determined using the solution (from step 2) considered optimal for degreasing the milk sample. Samples were contaminated with aliquots of 1, 5, 10, 25, and 50 larvae. For each milk sample (1.0 mL), 15 repetitions were analysed. The recovery of the larvae from the skim milk samples was higher (p = 0.0031) than that from the whole milk samples. No significant difference (p = 0.5681) was observed with regard to the percentage of recovered larvae when comparing the degreasing solutions. Nevertheless, the formalin-ether combination was more efficient for recovering the larvae (73.1%) than ether (71.9%), formalin (67.6%), and pure whole milk (70.0%). Concerning the limit of detection (using formalin-ether), all the samples contaminated with 5, 10, 25, and 50 larvae tested positive (minimum: 62.7%). Of the samples contaminated with a single larva, 66.7% tested positive. These results suggest that the centrifugation-sedimentation technique may be useful for recovering larvae of Toxocara spp. in naturally or experimentally contaminated milk samples obtained from a wide range of animal species. Key words: Toxocariasis, recovery of larvae, diagnosis ResumoO objetivo do estudo foi avaliar o limite de detecção de larvas de Toxocara canis em leite bovino comercial contaminado experimentalmente, a partir de uma técnica de centrifugo-sedimentação. Primeiramente, amostras de leite bovino comercial (integral e desnatado) foram contaminadas com 50 larvas de T. canis, para avaliação da interferência da gordura sobre a recuperação das larvas. Em uma segunda etapa, foi avaliada a ação de formalina 10% (100 µL), éter (100 µL) e combinação das soluções. A terceira etapa consistiu da verificação do limite de detecção de larvas, com uso da melhor solução desengordurante (etapa 2), em amostras de leite (1,0 mL) contendo 1; 5; 10; 25 e 50 larvas. Para cada análise de detecção do leite (1,0 mL), foram realizadas 15 repetições. Foi observado que o percentual de recuperação de larvas no leite desnatado foi significativamente maior (p= 0,0031) que o observado no leite integral. Na comparação das soluções, não houve diferença significativa (p= 0,5681) no percentual de larvas recuperadas. Entretanto, houve uma maior recuperação quando do emprego da combinação de formol-éter (73,1%) em relação ao éter (71,9%), ao formol (67,6%) e ao leite integral puro (70,0%). Em relação ao limite de detecção, com uso de formalina-éter, todas as alíquotas apresentaram resultado positivo (mínimo de 62,7% de larvas ...

Molecular and microscopic detection of natural and experimental infections of Toxocara vitulorum in bovine milk

“…Our data on the experimental contamination and detection of T. vitulorum larvae in milk samples are in close agreement with that previously recorded with other Toxocara species, specifically to name, T. canis [32]. The only difference is that this later study compared the effects of different degreasing solutions on milk samples and whether milk was whole or skimmed, factors which they found had no significant effects on the detection limits [32].…”

“…Formation of larvae aliquots followed by the experimental contamination of milk samples with different doses of larvae was done according to [32], but with minor modifications. Essentially, the recovered T. vitulorum larvae were counted on the custom microscope slide (sectioned into 4-mm squares) under the 4X of the optical microscope to obtain a count of #larvae/ml.…”

“…To recover larvae from milk samples, samples were subjected to the formalin-ether technique as described by [20,32,36], but with the minor modification of using chloroform instead of ether in the milk degreasing process. After the final centrifugation step, 200 μL aliquots from sediment of different larvae preparations were collected.…”

“…solution is considered as an easy, cheap and non-time wasting technique with the minimum detection limit of one larva of T. canis in milk [32]. Currently, DNA techniques have been widely used in the identification and genetic differentiation of T. vitulorum and other Toxocara species [40,41].…”

“…Thus diagnostic method/methods are essentially required for the proper detection of larvae of Toxocara species in milk, since minimizing the risk of the galagtogenic transmission. Up to date, only two reports dealing with detection of larvae of T. canis in the milk were published, with one reported on the detection limit of larvae in experimentally contaminated bovine milk [32], while the other on detecting larvae in the milk of experimentally infected rabbit as a paratenic host [20], with both studies had applied the centrifuge-sedimentation technique as the concentration-detection method. However, neither of the two studies has dealt with the dissemination of larvae in the milk of naturally infected animals.…”