Abstract
Objectives
The dehydrating power of cat litters may reduce the vitality of first-stage larvae (L1s) of feline lungworms, limiting copromicroscopical diagnosis. This study assessed the effect of the most commonly used cat litters on Aelurostrongylus abstrusus L1 survival.
Methods
Four types of cat litters were used: clumpling clay (group A); non-clumpling clay (group B); silica crystals (group C); and biodegradable (group D). A control group without litter (group E) was also included. On study day 0 (T0), L1s were obtained by the Baermann–Wetzel technique from the faeces of a naturally infected cat and ~100 larvae were injected in each of the 20 lungworm larvae-free faecal samples (~2 g each). Thereafter, four faecal samples per group were transferred into plastic cups containing the four different types of cat litters, or into empty cups (group E). The survival of L1s was assessed in each group after 3 (T3), 6 (T6), 12 (T12) and 24 (T24) h, using the Baermann–Wetzel technique.
Results
A decreasing trend of L1 survival was observed in all groups, with highest significant values at T0 compared with T3, T6, T12 and T24 (P <0.001). However, at T24, a significantly higher number (P <0.05) of L1s was extracted from faeces of the control group compared with the four groups with cat litters.
Conclusions and relevance
This study demonstrates how the survival of A abstrusus L1s, and therefore diagnosis, may be negatively influenced by the litter. The effect is time dependent, with a reduction in the number of vital larvae according to the type of litter, over time. False-negative results may be obtained, especially in cases of low parasitic load or when the sample is collected many hours after the emission.
Introduction
Aelurostrongylus abstrusus (Railliet, 1898; Strongylida, Angiostrongylidae) is a metastrongyloid nematode responsible for cardiorespiratory disease in cats and wild felids.1–4 The parasite has been reported in several European countries, with an apparent increasing distribution into previously non-endemic areas.1,5 According to a recent epidemiological survey conducted at European scale, feline lungworms are the second most frequent group of nematodes diagnosed in cats, and A abstrusus is the species most frequently detected. 1
This metastrongyloid is characterised by an indirect life cycle with adult parasites residing in the alveolar ducts and terminal respiratory bronchioles of the felid definitive host where females lay embryonated eggs hatching to first-stage larvae (L1s). L1s ascend the lung airways to the pharynx, they are swallowed and excreted with cat faeces in the environment, where they may penetrate snail intermediate hosts, eventually moulting into infective third-stage larvae (L3s).2,6 Insects, rodents, birds, amphibian and reptiles may act as paratenic hosts by ingesting infected gastropods.7,8 The definitive host acquires the infection by ingestion of intermediate or paratenic hosts, harbouring infective L3s. 6 Alternative routes of transmission have been proposed, including vertical transmission. 9 Additionally, the shedding of L3s in the environment by dead or alive gastropods, 10 and the snail-to-snail transmission of L3s (named ‘intermediesis’) 11 suggest that the feline aelurostrongylosis could also occur via the ingestion of L3 released in the environment.
The definitive diagnosis is achieved by the identification of L1s using the Baermann–Wetzel technique based on the positive hydrotropism of live larvae.12–14 However, this method is seldom employed in daily practice in Europe. 1
False-negative results may occur during the pre-patent period and due to the intermittent larval shedding in the faeces, which is a common feature of metastrongyloid lungworms. Owned cats often emit their faeces in cat litters, which usually have variable characteristics and, possibly, a different effect on the survival of larvae emitted with faeces. For example, the survival and sporulation of oocysts of Toxoplasma gondii and the viability of Tritrichomonas foetus is negatively influenced by cat litters.15,16 Even though the resistance of A abstrusus L1s in the environment has been described,6,17 and their survival at different temperatures has been demonstrated under laboratory conditions, 18 the effect of cat litter on the viability of A abstrusus L1s has not been investigated so far. Therefore, the objective of the present study is to evaluate the survival of A abstrusus L1s in faeces exposed to the most commonly used cat litters within a 24 h period.
Materials and methods
Larval identification and collection
The faeces of a donor cat were used as source for L1s of A abstrusus. The L1s were recovered using the Baermann–Wetzel technique and metastrongyloid larvae were identified to species-level using morphometrical keys.6,19 Soon after the isolation, the L1s were washed in saline solution, counted and split into vials containing ~100 larvae each.
Experimental design
The faeces emitted by a lungworm non-infected cat were collected daily, homogenised and divided in 20 aliquots of 2 g each. The aliquots of faeces were then injected with 100 L1s each (T0) and transferred into plastic cups containing the four different types of cat litters: clumping clay (group A); non-clumping clay (group B); silica crystals (group C); biodegradable (group D). For each group, four cups were used and a control group (group E) formed by four empty cups without litter was also employed. Cups containing cat litters were filled to 75% of the capacity and the faecal sample was gently covered with the litter. All the cups were stored in a room in which temperature and humidity ranged from 20°C to 23°C and 45% to 48%, respectively.
The vitality of L1s in the faecal samples was assessed after 3 (T3), 6 (T6), 12 (T12) and 24 (T24) h. At each time point, one faecal sample per group was collected, weighed and analysed by the Baermann–Wetzel technique. 13 Briefly, the faecal sample, wrapped on a double-layer gauze, was placed into the Baermann funnel filled with 150–200 ml warm tap water. After 24 h, the liquid was collected into a 50 ml tube and spun at 2500 rpm (600 g) for 5 mins. Thereafter, the supernatant (about 45 ml) was removed and the whole sediment was transferred on slides and microscopically observed (× 100) for the presence of L1s. For each sample, the total amount of live (ie, moving and/or not degenerated) larvae was registered, and L1 survival was estimated as the difference between the parasitic load injected at T0 and the number of live larvae recorded at each follow-up.
Statistical analysis
Two-way ANOVA for repeated measures was applied in order to evaluate the effect of different types of cat litters and time on the number of live A abstrusus L1s and on the weight of faecal samples. When significant differences were found Bonferroni’s post-hoc comparison was applied. Persons’ test and linear regression model were performed to assess significant correlation between the weight values of faeces and the number of live A abstrusus L1s in each group during the study period. Significance was set as P <0.05. Statistical analyses were performed using GraphPad Prism version 6.00 (GraphPad Software).
Results
The number of live A abstrusus L1s extracted in each experimental group during the study period is shown in Table 1. A statistically significant effect of the number of A abstrusus L1s was observed according to the cat litter and time (Figure 1). In particular, a higher number (P <0.05) of L1s was found in the control group (group E) with respect to the experimental groups (groups A–D) at T24. A decreasing trend of L1s was registered in all groups, with higher statistically significant values at T0 with respect to other time points (P <0.001). Group C showed the lower number of L1s at T6 and T24 with respect to T3, whereas groups A and B showed lower values at T24 with respect to T3 (P <0.05).
Number of alive Aelurostrongylus abstrusus first-stage larvae (L1s) recorded in each group
Group A = clumping clay; group B = non-clumping clay; group C = silica crystals; group D = biodegradable; group E = control without litter; T0 = time of L1s injection; T3 = 3 h after injection with L1s; T6 = 6 h after injection with L1s; T12 = 12 h after injection with L1s; T24 = 24 h after injection with L1s
Trend of Aelurostrongylus abstrusus first-stage larvae (L1s) recorded in each group (group A = clumping clay; group B = non-clumping clay; group C = silica crystals; group D = biodegradable; group E = without litter used as control) at L1 injection time (T0) and after 3 (T3), 6 (T6), 12 (T12) and 24 (T24) h
The faecal weight values measured in each group throughout study period are summarised in Table 2. Despite a statistically significant decreasing trend (P <0.05) of the faecal weight found in each group throughout the study period, no effect of the cat litters was found (Figure 2). A positive correlation was found between the weight of faeces and the number of A abstrusus L1s belonging to groups A–D, as demonstrated in a linear regression model, whereas any significant correlation was found in Group E (Figure 3).
Weight of faecal samples assessed at the time of injection of Aelurostrongylus abstrusus L1s (T0) and after 3 (T3), 6 (T6), 12 (T12) and 24 (T24) h in each group
Group A = clumping clay; group B = non-clumping clay; group C = silica crystals; group D = biodegradable; group E = control without litter
Trend of faecal weight values of each group (group A = clumping clay; group B = non-clumping clay; group C = silica crystals; group D = biodegradable; group E = without litter used as control) assessed at the time of first-stage larvae (L1s) injection (T0) and after 3 (T3), 6 (T6), 12 (T12) and 24 (T24) h
Significant Pearson’s correlation results and linear regression between faecal weight values and number of Aelurostrongylus abstrusus first-stage larvae (L1s) recorded in each group (group A = clumping clay; group B = non-clumping clay; group C = silica crystals; group D = biodegradable; group E = without litter used as control) at the time of L1s injection (T0) and after 3 (T3), 6 (T6), 12 (T12) and 24 (T24) h
Discussion
This survey represents the first investigation of the effect of different commercially available cat litters on the survival of A abstrusus L1s excreted in cat faeces. The results of this study demonstrate how the viability of L1s is negatively influenced by the contact of faeces with the cat litter, irrespective of their typology.
Previous studies demonstrated how the survival and sporulation of oocysts of T gondii may be influenced by different types of cat litters, with a reduction of infectivity of oocysts after 2–3 days of incubation of the faecal samples in the litters, whereas a longer permanence of the faecal samples (ie, ⩾5 days) was often lethal for oocysts. 15 The ability of T foetus to survive in different substrates, including distilled and tap water, cat urine, cat food and cat litter was also assessed, demonstrating that no trophozoites survived after 5 mins of exposure to the litter, which, otherwise, remain viable up to a maximum of 3 h in cat food and urine. 16
The reduction in the number of viable larvae observed in the current study was time dependent and reflects the progressive dehydration of samples exposed to the litter. Dehydration was more evident in the litter groups than in the control group, with a reduction of about 35% in the weight of faecal samples at T3 and up to 50% at T24, according to the type of litter. Consequently, the progressive dehydration produced a reduction of about 80% of the viable larvae after 3 h and up to 100% after 24 h. The findings obtained in the current survey emphasise how the time of faecal samples spent in the litter negatively affects the viability of L1s and, consequently, it may compromise diagnosis leading to false-negative results from the Baermann–Wetzel extraction.
The faecal samples used in this study were infected with about 100 larvae. This number is larger than that generally observed in asymptomatic adult animals, 20 meaning that false-negative results could be expected even before 24 h in the case of low-parasitic load faecal samples. However, this study was carried under controlled laboratory conditions, which may make some factors different from those occurring under natural settings. For example, cats do not always cover faeces with litter, and the dehydrating power of litter may be reduced by the presence of urine and by the different consistencies of the faecal matter. These factors may slightly prolong the survival of L1s.
Conclusions
The permanence of faecal samples on cat litter may compromise the diagnosis of aelurostrongylosis, leading to false-negative results, especially when the parasitic load is low and/or when the sample is collected many hours after the emission.
On the basis of the cat’s attitude and clinical signs, veterinarians should always include verminous bronchopneumonia in the differential diagnosis, and possibly analyse more fresh faecal samples emitted on consecutive days in order to increase the sensitivity of the Baermann–Wetzel sedimentation diagnosis.
Footnotes
Conflict of interest
The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Funding
The authors received no financial support for the research, authorship, and/or publication of this article.
